Bioactive botanical cosmetic compositions and processes for their production

ABSTRACT

The present invention is directed to bioactive botanical cosmetic compositions derived from membrane and cell serum fractions of plant cell juice. The present invention also relates to the methods for preparing these bioactive botanical cosmetic compositions and the uses of these compositions in various cosmetic formulations and as topical skin cosmetic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/116,924, filed May 7, 2008, which is a divisional of U.S.patent application Ser. No. 10/351,910, filed Jan. 24, 2003, now U.S.Pat. No. 7,442,391, issued Oct. 28, 2008, which claims the prioritybenefit of U.S. Provisional Patent Application Ser. No. 60/351,886,filed Jan. 25, 2002, the disclosures of which are hereby incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to bioactive botanical cosmeticcompositions and processes for their production and their use.

BACKGROUND OF THE INVENTION

Over the past several decades, the cosmetic industry has embraced theuse of plants and plant products in a variety of cosmetic formulationsand products. Although this trend is expected to continue, there is aneed for more refined and higher quality botanical ingredients thatconsistently exhibit characteristics that are appealing to the cosmeticindustry and consumers. Some of these appealing bioactivecharacteristics include anti-inflammatory and antioxidant activity.Coloration, safety, compatibility, and increased shelf life are alsovaluable characteristics of cosmetic formulations derived from botanicalingredients.

The cosmetic industry as a whole has increased its support of efforts todevelop and market “natural” cosmetic formulations using a host ofsingle and blended botanical ingredients that are currently available tothe industry. This approach differs from the synthetic ingredient-basedapproach that has allowed the cosmetic industry to develop cosmeticswith consistent product integrity, performance, and shelf life of rawmaterial ingredients. One of the major deterrents toward the use ofbotanical ingredients is the inconsistency of the performance andstability of the ingredients, especially with regard to bioactivebotanical ingredients. Many of the bioactive botanical cosmeticingredients now used as ingredients in cosmetic formulations exhibitlost potency, odor deviations, unwanted darkening in coloration, andundesirable sedimentation. These negative attributes increase the riskof microbiological contamination and proliferation, instability, andsafety concerns with regard to the final products made from thebioactive botanical ingredients.

In order to ensure quality, safety, and consistency, the cosmeticindustry has developed and implemented various standard operatingprocedures and strict specification controls for all incoming rawmaterials for use in cosmetic formulations. Most, if not all, of thecurrent botanical extracts fail to comply with the increasing controlsand consistency parameters of the cosmetic industry. Current plantextraction methods limit product specification parameters leaving manywindows of variability for quality, performance, and compatibility. Inaddition, current extraction methods fail to deliver the full spectrumof activities that exist within plant cells. Thus, the full potential ofbotanical-based cosmetic formulations is not being realized due to theinadequacy of the extraction methods for bioactive botanical cosmeticingredients.

Many of the current methods for extracting bioactive components fromplants involve techniques that are harmful to the plant tissue or thebioactive components of interest contained in that tissue, or both.Further, many of the current extraction and separation methods yieldcrude botanical extracts that contain biological or chemicalcontaminants that can cause a loss of bioactivity potency, increasedcytotoxicity, and decreased shelf life. Further, in order to yield morerefined botanical extracts, current extraction methods often require theuse of harsh chemical solvents.

Thus, there is a need for a method of extracting bioactive botanicalcompositions that preserves the bioactivity of the composition and thatyield consistent results from lot-to-lot. Further, botanicalcompositions that are able to meet the industry standards with respectto shelf life, cytotoxicity, quality, and performance are needed in thecosmetic industry.

The present invention is directed to overcoming these deficiencies inthe art.

SUMMARY OF THE INVENTION

The present invention relates to a bioactive botanical cosmeticcomposition including (1) a membrane fraction derived from cell juiceextracted from a fresh plant biomass and (2) a stabilizing agent. Themembrane fraction has antiproteolytic activity, cell growth inhibitionactivity, and/or both antiproteolytic and cell growth inhibitionactivities. The antiproteolytic activity is due to inhibition of atleast one proteinase and the cell growth inhibition activity is due toinhibition of proliferation of at least one type of cell.

The present invention also relates to a bioactive botanical cosmeticformulation suitable for topical application to a mammal. The bioactivebotanical cosmetic formulation includes a cosmetically acceptablecarrier and a cosmetically effective amount of the bioactive botanicalcosmetic composition described above.

The present invention also relates to a method for inhibitinganti-inflammatory activity in skin tissue of a mammal. This methodinvolves applying to the skin tissue the above described bioactivebotanical cosmetic composition in an amount effective to enhance theantiproteolytic activity in the skin tissue.

The present invention also relates to a method for normalization of celldisorders in skin tissue of a mammal. This method involves applying tothe skin tissue the above-described bioactive botanical cosmeticcomposition in an amount effective to inhibit unwantedhyper-proliferation of skin cells.

The present invention also relates to a method for preparing a bioactivebotanical cosmetic composition, which involves providing a plant celljuice that has been extracted from a fresh plant biomass. The plant celljuice is then treated under conditions effective to separate it into amembrane fraction and a cell juice supernatant. The membrane fraction istransformed under conditions effective to yield a stable bioactivebotanical cosmetic composition exhibiting antiproteolytic, cell growthinhibition activity, and/or both antiproteolytic and cell growthinhibition activities, where the antiproteolytic activity is due toinhibition of at least one proteinase and the cell growth inhibitionactivity is due to inhibition of cell growth of at least one type ofcell.

The present invention also relates to a bioactive botanical cosmeticcomposition made by the method described immediately above.

The present invention also relates to a bioactive botanical cosmeticformulation suitable for topical application to a mammal. Theformulation includes a cosmetically acceptable carrier and acosmetically effective amount of the bioactive botanical cosmeticcomposition described immediately above.

The present invention also relates to a method for inhibitinganti-inflammatory activity in skin tissue of a mammal by applying to theskin tissue the bioactive botanical cosmetic composition described abovein an amount effective to enhance the antiproteolytic activity in theskin tissue.

The present invention further relates to a method for normalization ofcell disorders in skin tissue of a mammal, involving applying to theskin tissue the bioactive botanical cosmetic composition having cellgrowth inhibition activity in an amount effective to inhibit unwantedhyper-proliferation of skin cells.

The present invention also relates to a bioactive botanical cosmeticcomposition including the membrane fraction made by the method describedabove.

The present invention also relates to a bioactive botanical cosmeticcomposition including (1) a cell serum fraction derived from cell juiceextracted from a fresh plant biomass, where the cell serum fraction hasantioxidant activity, cell growth stimulation activity, and/or bothantioxidant and cell growth stimulation activities, and (2) astabilizing agent. The cell growth stimulation activity is due tostimulation of proliferation of at least one type of cell.

The present invention also relates to a bioactive botanical cosmeticformulation suitable for topical application to a mammal, including acosmetically acceptable carrier and a cosmetically effective amount ofthe bioactive botanical cosmetic composition described immediatelyabove.

The present invention further relates to a method for enhancing theantioxidant activity in skin tissue of a mammal, involving applying tothe skin tissue of the mammal the bioactive botanical cosmeticformulation described above in an amount effective to increase theantioxidant activity in the skin tissue.

The present invention also relates to a method for stimulation of cellproliferation in skin tissue of the mammal, involving applying to theskin tissue the bioactive botanical cosmetic formulation described abovein an amount effective to stimulate fibroblast proliferation in the skintissue.

The present invention also relates to a method for preparing a bioactivebotanical cosmetic composition, which involves providing a plant celljuice that has been extracted from a fresh plant biomass. The plant celljuice is then treated under conditions effective to separate the plantcell juice into a membrane fraction and a cell juice supernatant. Thecell juice supernatant is processed under conditions effective toseparate the cell juice supernatant into a cytoplasm fraction and a cellserum fraction. The cell serum fraction is refined under conditionseffective to yield a cell serum fraction filtrate. The cell serumfraction filtrate is stabilized under conditions effective to yield astable bioactive botanical cosmetic composition exhibiting antioxidantactivity, cell growth stimulation activity, or both antioxidant and cellgrowth stimulation activities.

The present invention also relates to a stable bioactive botanicalcosmetic composition made by the method described immediately above.

The present invention also relates to a bioactive botanical cosmeticformulation suitable for topical application to a mammal, including acosmetically acceptable carrier and a cosmetically effective amount ofthe bioactive botanical cosmetic composition made by the processdescribed above.

The present invention also relates to a method for enhancing theantioxidant activity in skin tissue of a mammal. This method involvesapplying to the skin tissue the bioactive botanical cosmetic compositiondescribed above in an amount effective to increase the antioxidantactivity in the skin tissue.

The present invention further relates to a method of stimulation of cellproliferation in skin tissue of a mammal. This method involves applyingto the skin tissue the bioactive botanical cosmetic compositiondescribed above in an amount effective to stimulate fibroblastproliferation in the skin tissue.

The method for preparing bioactive botanical cosmetic compositions isadvantageous over the methods currently available in that it yieldsplant extracts that capture the full spectrum of activity contained inthe plant cells. These extracts can then be separated into either cellserum or membrane components, while still maintaining the bioactivitycontained within each component. Further, the compositions producedaccording to the method of the present invention have cytotoxicityprofiles that are demonstrably safer for skin than other conventionalplant extracts. In addition, the compositions of the present inventionmeet the microbial requirements of the cosmetic industry. Thus, due tothe consistency, quality, safety, shelf life, and significantbioactivity potency with regard to anti-inflammatory and antioxidantcapabilities, the bioactive botanical cosmetic compositions of thepresent invention are significant improvements over the botanicalcosmetic ingredients available currently.

The bioactive botanical cosmetic compositions of the present inventionexhibit the anti-inflammatory and antioxidant activities that arevaluable to the cosmetic industry. Further, the cytotoxicity profiles ofthe bioactive botanical cosmetic compositions are within the industrystandards for cosmetic ingredients and exhibit cell proliferativestimulatory activity and certain cell growth inhibitory activity atlevels that are advantageous as topical skin cosmetics. The method forpreparing the bioactive botanical cosmetic ingredients of the presentinvention may be used on a wide variety of plants to yield consistent,stable, and quality bioactive botanical cosmetic compositions.

The bioactive botanical cosmetic compositions of the present inventionmeet the industry standards with respect to the microbial requirementsof cosmetic raw material ingredients. The industry standard requiresthat all active and inactive ingredients (i.e., all excipients of thecosmetic formulations) not be such that they contribute to the finishedformulation composition of a cosmetic product. Typically, these finishedformulation compositions have preservative systems that preventmicrobial contamination that could risk the integrity of the product. Inone standard use by the industry to test the protective strength of apreservative system, a product is subjected to a 28-day challenge testduring which time microorganisms are inoculated into a product to see ifit withstands these treatments without becoming contaminated. Inaddition, in the cosmetic industry, each ingredient is also scrutinizedto make sure that the level of microorganisms is not so high as toresult in subsequent contamination of a product or pose a risk on theshelf life of the product if it is not optimally preserved.

Specifically, the industry standard statistic that “the microbiologicalrequirements for active ingredients in the cosmetic area state that atotal microbial count of a maximum 100 microorganisms per gram or per mlmay be tolerated. The sample (10 g) must furthermore be free fromEscherichia coli, Candida albicans, Pseudomonas sp., and Staphylococcusaureus” (G. A. Nowak, “Cosmetic Preparations,” Verlag fur Chem.,Augsburg, 1:126 (1985), the entire disclosure of which is incorporatedherein by reference). The bioactive botanical cosmetic compositions ofthe present invention satisfy the above requirements and therefore poseno risk to finished cosmetic formulation compositions.

The bioactive botanical cosmetic compositions of the present inventionhave highly valuable bioactive attributes with respect to the skin,including, for example, anti-inflammatory and antioxidant activities, aswell as cell proliferative stimulatory characteristics. It is generallyknown that there is the balance between newly born and dead skin cells.Optimum attributes of skin are found in young and healthy skin (i.e.,usually found in people under the age of 25). Before this age, skincells are in a regulated state and are in a well-balanced system ofrenewal; born at the deepest basal layers and eventually proliferating(i.e., rising from the deep skin) to the top (i.e., the layer which wevisually appreciate). This balance of cells being shed is part of anequilibrium of renewal. This equilibrium is lost as a result of adultaging, and there is a slow down in the proliferation rate after newcells are born. The concept of increasing or stimulating cellproliferation is based on restoring the optimum equilibrium that isfound in “younger” skin. This has led to an interest in cellproliferation stimulators such as retinoids and AHA (alpha hydroxyacids). Such ingredients basically increase the rate of proliferationthrough an irritation mode of action, leading to smoother,younger-looking skin due to accelerated cell proliferation. Thebioactive botanical cosmetic compositions of the present invention, inparticular those derived from the cell serum fractions, exhibit abilityto stimulate cell proliferation.

In a more comprehensive manner, increased skin proliferation is a key towound healing and dermatological conditions. There are certaindermatological conditions whereby skin proliferation tends to be in ahyper-proliferative state. These conditions border and cross diseasestates manifesting themselves on the skin. Conditions such as psoriasis,eczema, and dandruff are all hyper-proliferative conditions. Skin cellgrowth inhibitors are then the obvious and suggested approach to slowingdown the rate of proliferation to normalize the rate. Various of thebioactive botanical cosmetic compositions of the present invention, inparticular those derived from the membrane fraction, exhibit such cellinhibition attributes.

Inflammation occurs for many reasons on the skin. Usually associatedwith injury, today experts are beginning to understand the cascadingeffects of micro-inflammation. This micro-inflammation of the skin canresult from irritating ingredients such as soaps and cytotoxicingredients, ordinary UV light such as minimal sunlight, and in a moredrastic manner from intense exposure to the sun. Recently, the role ofinflammation on skin aging has been more clearly understood andsuggested to be an indirect route to formation of free-radicals, whichhave been clearly implicated for their role in membrane lipid oxidation.Thus, anti-inflammatory agents are important cosmetic ingredients, butthe regulatory restrictions limit their use as drugs. However, thebioactive botanical cosmetic compositions of the present invention,particularly those derived from the membrane fraction, demonstrateanti-inflammatory attributes.

The role of antioxidants has become increasingly important for nutritionand cosmetic products. Antioxidants retard, protect against, and helprepair the adverse effects of oxidative degradation. In the plant world,nature has provided natural antioxidants that protect against manyoxidative factors. The bioactive botanical cosmetic compositions of thepresent invention, particularly those derived from the cell serumfraction, demonstrate such antioxidant activity.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic drawing demonstrating one embodiment of theprocess for preparing the bioactive botanical cosmetic compositions ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to bioactive botanical cosmeticcompositions derived from either the membrane fraction or the cell serumfraction of plants. As used herein, the term “Membrane-Derived CosmeticComposition” generally refers to a bioactive botanical cosmeticcomposition of the present invention that is derived from the membranefraction of a plant. The term “Serum-Derived Cosmetic Composition”generally refers to a bioactive botanical cosmetic composition of thepresent invention that is derived from the cell serum fraction of aplant.

The present invention also relates to processes for producing thebioactive botanical cosmetic compositions of the present invention, aswell as methods for using the compositions.

Membrane-Derived Cosmetic Compositions

The Membrane-Derived Cosmetic Compositions of the present inventioninclude (1) a membrane fraction derived from cell juice extracted from afresh plant biomass and (2) a stabilizing agent. The membrane fractionhas antiproteolytic activity, cell growth inhibition activity, and/orboth antiproteolytic and cell growth inhibition activities. Theantiproteolytic activity is due to inhibition of at least one proteinaseand the cell growth inhibition activity is due to inhibition ofproliferation of at least one type of cell. Examples of stabilizingagents that are suitable for use in the present invention includeemulsifiers, preservatives, antioxidants, polymers, and mixturesthereof.

In one aspect of the present invention, the Membrane-Derived CosmeticComposition has antiproteolytic activity against proteinase groups suchas serine proteinases and matrix metalloproteinases. Examples of theserine proteinase include neutrophil elastase and trypsin inhibitor. Anexample of a matrix metalloproteinase is gelatinase B. In another aspectof the present invention, the inhibition of the proteinase isreversible. Since serine proteinases have certain positive physiologicalroles when present at controlled levels, use of reversible inhibitorswill not impact these normal enzymatic functions. The reversibleinhibition would not cause undesirable long term modifications todefense and repair mechanisms which can be impacted by irreversibleinhibitors.

The Membrane-Derived Cosmetic Composition of the present invention hasan antiproteolytic potency ranging from an IC₅₀ value of between about0.1 and about 25.0 μg dry matter/ml. As used in the present application,the term “IC₅₀ value” represents the concentration of dry mattercontained in the membrane fraction required to achieve 50 percentinhibition of the proteinase.

The Membrane-Derived Cosmetic Composition of the present invention has acell growth inhibition activity potency ranging from an NRU₅₀ value ofbetween about 25 and 500 μg dry matter/ml. As used in the presentapplication, the term “NRU₅₀ value” represents the concentration of drymatter in the membrane fraction required to reduce the viability of thetype of cell to 50 percent. An example of a type of cell that isinhibited from proliferating due to the Membrane-Derived CosmeticComposition is a fibroblast.

The Membrane-Derived Cosmetic Composition of the present invention maybe derived from membrane fractions of all types of plants. Examples ofsuitable plants that may be used as sources of fresh plant biomass inthe present invention include plants from the following families:Asteraceae, Fabaceae, Lamiaceae, and Poaceae. In particular, examples ofspecific plants that have been tested and found appropriate as freshplant biomass sources include, without limitation, Trifolium pratense,Nelumbo nucifera, Calendula officinalis, Medicago sativa, Lavandulaangustifolia, Salvia officinalis, and Hordeum vulgare. TheMembrane-Derived Cosmetic Composition may be derived from flower tissue(e.g., Trifolium pratense, Nelumbo nucifera, Calendula officinalis)and/or from leaf and stem tissue (e.g., Trifolium pratense, Nelumbonucifera, Salvia officinalis) of plants.

In one embodiment, the membrane fraction derived from the plant celljuice makes up between about 0.5 and about 95 weight percent of theMembrane-Derived Cosmetic Composition.

The Membrane-Derived Cosmetic Composition of the present invention canhave the following specific physico-chemical values: (1) a non-volatileresidue value of between about 0.1 and 30 percent; (2) a specificgravity value of between about 0.5 and 2.0 g/cm³; (3) a viscosity valueof between about 300 and 50,000 cps; and (4) a pH value of between about2.5 and 9.5.

The present invention also relates to a bioactive botanical cosmeticformulation suitable for topical application to a mammal, including tohumans, where the formulation includes a cosmetically acceptable carrierand a cosmetically effective amount of the Membrane-Derived CosmeticComposition. Examples of suitable cosmetically acceptable carriers foruse in the present invention include a hydrophilic cream base, ahydrophilic lotion base, a hydrophilic surfactant base, a hydrophobiccream base, a hydrophobic lotion base, and a hydrophobic surfactantbase. In one embodiment of the formulation, the Membrane-DerivedCosmetic Composition is present in an amount ranging from between about0.001 percent and about 90 percent of the total weight of theformulation.

The present invention also relates to a method for inhibitinganti-inflammatory activity in skin tissue of a mammal, which methodinvolves applying to the skin tissue the Membrane-Derived CosmeticComposition in an amount effective to enhance the antiproteolyticactivity in the skin tissue.

The present invention also relates to a method for normalization of celldisorders in skin tissue of a mammal. This method involves applying tothe skin tissue the Membrane-Derived Cosmetic Composition in an amounteffective to inhibit unwanted hyper-proliferation of skin cells.

Serum-Derived Cosmetic Compositions

The Serum-Derived Cosmetic Compositions of the present invention include(1) a cell serum fraction derived from cell juice extracted from a freshplant biomass, where the cell serum fraction has antioxidant activity,cell growth stimulation activity, and/or both antioxidant and cellgrowth stimulation activities, and (2) a stabilizing agent. The cellgrowth stimulation activity is due to stimulation of proliferation of atleast one type of cell. Examples of stabilizing agents suitable for usein the present invention include a preservative and an antioxidant.Suitable preservatives for use in the present invention includepotassium sorbate, sodium benzoate, sodium methyl paraben, and citricacid. An example of a suitable antioxidant for use in the presentinvention is sodium metabisulfite.

In one embodiment, the antioxidant activity of the Serum-DerivedCosmetic Composition includes superoxide scavenging activity andneutrophil respiratory burst inhibitory activity. The Serum-DerivedCosmetic Composition has a superoxide scavenging potency ranging from anICR₅₀ value of between about 50 and 190 μg of dry matter/ml. As used inthe present application, the term “ICR₅₀ value” represents theconcentration of dry matter contained in the cell serum fractionrequired to inhibit 50 percent of cytochrome c reduction. The cellserum-derived cosmetic ingredient has a cell growth stimulation potencyranging from between about 1.0 and 125 μg of dry matter/ml and an NRUvalue of between about 110 and 190 percent, where the “NRU value”represents cell viability. The Serum-Derived Cosmetic Compositioninhibits the respiratory bursts at between about 1.0 and 5.0 μg drymaterial/ml and stimulates the respiratory bursts at between about 120and 180 μg dry material/ml. The Serum-Derived Cosmetic Composition hasthe ability to cause biphasic modulation of respiratory bursts fromphorbol myristate acetate-stimulated neutrophils.

An example of a type of cell that is stimulated to proliferate due tothe Serum-Derived Cosmetic Composition includes a fibroblast.

The Serum-Derived Cosmetic Composition of the present invention may bederived from cell serum fractions from all types of plants. Examples ofsuitable plants that may be used as sources of fresh plant biomass inthe present invention include plants from the following families:Asteraceae, Fabaceae, Lamiaceae, and Poaceae. In particular, examples ofspecific plants that have been tested and found appropriate as freshplant biomass sources include, without limitation, Trifolium pratense,Nelumbo nucifera, Calendula officinalis, Medicago sativa, Lavandulaangustifolia, Salvia officinalis, and Hordeum vulgare. The Serum-DerivedCosmetic Composition may be derived from flower tissue (e.g., Trifoliumpratense, Nelumbo nucifera, Calendula officinalis) and/or from leaf andstem tissue (e.g., Trifolium pratense, Nelumbo nucifera, Horedeumvulgare, Lavandula angustifolia, Medicago sativa, and Salviaofficinalis).

In one embodiment, the cell serum fraction derived from the plant celljuice makes up between about 1 and 10 weight percent of theSerum-Derived Cosmetic Composition.

The present invention also relates to a bioactive botanical cosmeticformulation suitable for topical application to a mammal, including acosmetically acceptable carrier and a cosmetically effective amount ofthe Serum-Derived Cosmetic Composition. Examples of suitablecosmetically acceptable carriers include, without limitation, ahydrophilic cream base, a hydrophilic lotion base, a hydrophilicsurfactant base, a hydrophobic cream base, a hydrophobic lotion base,and a hydrophobic surfactant base. In one embodiment, the Serum-DerivedCosmetic Composition is present in an amount ranging from between about0.001 percent and 95 percent of the total weight of the cosmeticformulation.

The present invention further relates to a method for enhancing theantioxidant activity in skin tissue of a mammal, involving applying tothe skin tissue the Serum-Derived Cosmetic Composition in an amounteffective to increase the antioxidant activity in the skin tissue.

The present invention also relates to a method for stimulation of cellproliferation in skin tissue of a mammal, involving applying to the skintissue the Serum-Derived Cosmetic Composition in an amount effective tostimulate cell proliferation in the skin tissue.

Overall Process for Preparing Bioactive Botanical Cosmetic Compositions

By way of example, the overall process for preparing the bioactivebotanical cosmetic compositions of the present invention is describedbelow in reference to FIG. 1. As depicted in FIG. 1, fresh plants areharvested, collected, and washed 2 to yield fresh plant biomass. Thisfresh plant biomass is subjected to grinding, maceration, and pressing 4to yield plant cell juice 6 and press-cake 8. Plant cell juice 6 is thenfiltered through nylon mesh 10 to yield filtered plant cell juice 12.Filtered plant cell juice 12 is exposed to microwave treatment 14 inorder to coagulate plant cell juice 12. The coagulated plant cell juiceis cooled 16 and then subjected to centrifugation 18 in order to yieldmembrane fraction 20 and plant cell juice supernatant 30. Membranefraction 20 is used to prepare membrane-derived bioactive botanicalcosmetic composition 28 (i.e., the Membrane-Derived CosmeticComposition), as described below. Plant cell juice supernatant 30 isused to prepare cell serum-derived bioactive botanical cosmeticcomposition 52 (i.e., the Serum-Derived Cosmetic Composition), asdescribed below.

To produce bioactive botanical cosmetic composition 28, membranefraction 20 is incorporated into polymer matrix 22 and stabilized withprepared polymers, preservatives, and antioxidants 24. The stabilizedmembrane fraction is then neutralized 26 to yield the membrane-derivedbioactive botanical cosmetic composition 28.

To produce bioactive botanical cosmetic composition 52, plant cell juicesupernatant 30 is subjected to isoelectric precipitation 32 to yield amixture containing cytoplasm fraction 36 and cell serum fraction 38. Inorder to separate cell serum fraction 38 from cytoplasm fraction 36, themixture is subjected to centrifugation 34. Cell serum fraction 38 isthen subjected to microwave treatment to cause coagulation 42. Dependingon the plant source, prior to microwave treatment, cell serum fraction38 is first pH-adjusted. After coagulation 42, the mixture is thencooled 44, followed by filtration 46 to yield cell serum filtrate 48.Cell serum filtrate 48 is stabilized with preservatives and antioxidants50 to yield cell serum-derived bioactive botanical cosmetic composition52.

Process for Preparing the Membrane-Derived Cosmetic Compositions

In one embodiment, the process for preparing the Membrane-DerivedCosmetic Compositions is as follows. This method involves providingplant cell juice that has been extracted from a fresh plant biomass. Theplant cell juice is then treated under conditions effective to separateit into a membrane fraction and a cell juice supernatant. The resultingmembrane fraction has antiproteolytic activity, cell growth inhibitionactivity, or both antiproteolytic and cell growth inhibition activities.The membrane fraction is then converted under conditions effective toyield a stable bioactive botanical cosmetic composition exhibitingantiproteolytic, cell growth inhibition activity, or bothantiproteolytic and cell growth inhibition activities, where theantiproteolytic activity is due to inhibition of at least one proteinaseand the cell growth inhibition activity is due to inhibition of cellgrowth of at least one type of cell.

The plant cell juice may be extracted from all types of plants. Examplesof suitable plants that may be used as sources of fresh plant biomass inthe present include, without limitation, plants from the followingfamilies: Asteraceae, Fabaceae, Lamiaceae, and Poaceae. In particular,examples of specific plants that have been tested and found appropriateas fresh plant biomass sources include, without limitation, Trifoliumpratense, Nelumbo nucifera, Calendula officinalis, Medicago sativa,Lavandula angustifolia, Salvia officinalis, and Hordeum vulgare. Variousparts of the plants may be used. For example, the stems and leaf tissuemay be used for many types of plants. For other plants, the flowers maybe used as sources of plant cell juice for use in the present invention.For example, one embodiment of the present invention uses flower tissueof Trifolium pratense, Nelumbo nucifera, or Calendula officinalis forthe extraction of the plant cell juice. In another embodiment, the leafand stem tissue of Trifolium pratense, Nelumbo nucifera, or Salviaofficinalis is used.

The plant cell juice may be extracted using various extractiontechniques. However, the extraction technique should result in plantcell juice that preserves the bioactive components of the plant.

An exemplary method of preparing the plant biomass for use in extractionof plant cell juice involves harvesting, collecting, and washing of thefresh plants. Suitable steps to follow for preparing the fresh plantbiomass include, for example, the following: (1) preservation of theinherent moisture content of the plant cells; (2) optimization of theheight of cut used during harvesting of above-ground plant tissue; (3)reservation of plant integrity during harvesting (e.g., during cuttingof the above-ground plant tissue); (4) minimization of environmentalimpact and time factors of biological degradation of the plant biomass;and (5) cleaning of the plant biomass prior to processing (e.g., priorto grinding and maceration). Each of these steps is discussed below.

Preservation of Inherent Moisture Content:

The cutting should be done to avoid wilting due to moisture loss.Optimal conditions are those where natural moisture content ismaintained and preserved.

Optimal and Preferred Height of Cut:

The plants should be cut at least several centimeters above the groundto limit the amount of soil and other debris in the collected biomass.For example, all useable leaf and stem biomass of any given plant source(e.g., alfalfa, barley, lavender, or sage) may be cut at a height ofgreater than or equal to 5 centimeters above ground. If flower tissue isused as the plant biomass source, the flowers are separated from thewhole plant prior to extraction of the plant cell juice.

Preservation of Plant Integrity During Harvesting:

Harvesting of the plant biomass may be by cutting the above ground stemand leaf tissue of the plant. The cutting is conducted in a manner thatavoids or minimizes the chopping, mashing, crushing, or other type ofinjury of the plant. For large-scale industrial harvesting, where it maynot be possible to avoid chopping due to the type of equipment required,care is taken to minimize injury that could lead to microbial growth,moisture loss, intensification of oxidation, polymerization,isomerization, and hydrolysis processes (i.e., unwanted catabolicprocesses) in collected plants. For example, in one embodiment of thepresent invention, lavender and sage are cut and collected by hand aswhole plants. In another embodiment, alfalfa and barley tissue are cutusing harvesting equipment. In that case, the minimum chopping heightabove ground for each plant is greater than or equal to 5 centimeters.Further, particular attention is made to minimize injury during andafter cutting. In another embodiment, marigold whole plants arecollected by hand and the flowers are then separated for furtherprocessing.

Minimization of Environmental Impact and Time Factors of Degradation:

Delivery time of cut plant material to the processing facility andexposure of biomass to sun, high temperature, and other negativeenvironmental factors, should be minimized to prevent the impact ofunwanted degradation processes as described above. For example, in oneembodiment of the present invention, the delivery time for alfalfa andbarley for further processing does not exceed 30 minutes from the timeof cutting. In another embodiment, plants that undergo long distancetransport are treated to a post-cutting procedure involving immediatelyplacing the plant biomass into Styrofoam coolers containing bags offrozen gel packs to help maintain freshness and natural moisture contentduring overnight delivery to the processing facility. These procedureswere conducted for plant biomass from lavender, marigold, and sage.Other post-cutting procedures that achieve the results described abovemay be used as well.

Cleaning Step Prior to Grinding and Maceration:

A washing step to remove the soil particles and other debris from plantsprior to further processing is performed once the plant tissue isharvested. The washing is achieved using a low-pressure rinse for ashort duration under conditions to prevent the initiation of the releaseof the cell juice from biomass, to cause injury, or to remove valuablecomponents. For example, in one embodiment of the present invention, thewashing of the plant biomass was accomplished in less than or equal to 5minutes with a water pressure of less than or equal to 1 kg/cm².Residual water wash did not contain any green or yellow pigments, whichindicates the absence of subsequent injury. The excess water is removedfrom washed biomass in order to keep the dry matter content close tonatural level.

After the plant tissue biomass is harvested, as described above, furtherprocessing of the plant tissue biomass is performed to yield plant celljuice. In one embodiment, the harvested plant tissue biomass issubjected to grinding, maceration, and pressing to extract theintracellular content, i.e., the cell juice, and to separate it from thefiber-enriched press-cake containing predominantly cell walls.

An example of a suitable processing protocol involves the stepsdescribed below. A hammer mill may be used to grind plants to yieldplant tissue particles of a small size in a short time and withoutsignificant increase of biomass temperature. In one embodiment, amodified hammer mill is used to produce the maximum size of maceratedplant particles less than or equal to 0.5 centimeters during less thanor equal to 10 seconds of treatment, where the increase of biomasstemperature is less than or equal to 5° C.

Exposure of ground and macerated plant biomass is minimized to preventthe impact of unwanted catabolic processes, as described above. Theextraction of the plant cell juice and its separation from thepress-cake is commenced as soon as possible after grinding andmaceration of the plant biomass. The plant biomass is processed in ashort time and without significant increase in temperature. In oneembodiment, immediately after grinding and maceration, the plant biomassis pressed using a horizontal, continuous screw press (Compact Press“CP-6”, Vincent Corporation, FL). The pressure on the cone is maintainedat level 24 kg/cm², screw speed is at 12 rpm, and the temperatureincrease is less than or equal to 5° C.

The initial cell juice usually contains small fiber particles, which canabsorb valuable cell juice components and also block the hoses andpumps. The above particles should be removed by filtration or low-speedcentrifugation. For example, the initial cell juices produced after thepressing step are filtered through four layers of nylon fabric prior tousing the plant cell juice in the methods of the present invention.

Once plant cell juice is extracted, the plant cell juice is then treatedto a processes involving (1) performing a “membrane fraction coagulationstep” to yield a coagulated cell juice mixture and (2) performing a“membrane fraction separation step” on the coagulated cell juice mixtureto yield a membrane fraction and a cell juice supernatant. In oneembodiment, the membrane fraction coagulation step includesdestabilizing the cell juice to yield a coagulated cell juice mixture.The destabilizing may be achieved using a variety of destabilizationtechniques, including, for example, temperature treatment,electro-membrane treatment, and chemical treatment. Suitable temperaturetreatment for use in the present invention may include (1) heating thecell juice extract to a treatment temperature required to inducecoagulation of the membrane fraction (e.g., to a temperature of betweenabout 45 and 70 degrees Celsius) and (2) cooling the cell juice to atemperature effective to allow further quantitative separation of saidmembrane fraction from said cell juice supernatant (e.g., to atemperature of between about 30 and 45 degrees Celsius). Afterdestabilization is achieved, a membrane fraction separation step isperformed. This step includes, for example, separating the coagulatedcell juice mixture into the membrane fraction and the cell juicesupernatant using separating techniques including filtration andcentrifugation.

The freshly obtained membrane fraction commonly referred to in the art,as “protein-vitamin concentrate,” is a paste having intensive color andspecific odor that is plant raw material source specific. The membranefraction is represented predominantly by chloroplasts present in thegreen parts of plant or mostly by chromoplasts present in flowers. Thecomposition of the membrane fraction includes predominantlyphospholipids, membrane proteins, chlorophyll, and carotenoids. Thedrying of membrane fraction results in irreversible loses of manyvaluable properties required for the exploration of membrane fraction asa cosmetic ingredient. Without drying, the unstable membrane fraction isquickly transformed into the dark color un-dispersible and insolubleconglomerates having strong and non-characteristic odor. As result, suchmaterial cannot be used as a cosmetic ingredient. The describedprocedure that follows allows for transformation of freshly obtainedmembrane fractions into stable and active cosmetic ingredients.

Once the membrane fraction is separated from the cell juice supernatant,the membrane fraction is then subjected to a formulation process priorto aggregation of the membrane fraction, including the following steps:(1) performing a “stabilization step” to yield a stabilized membranefraction component; (2) performing a “polymer matrix incorporation step”on the stabilized membrane fraction component to yield a membranefraction matrix; and (3) performing a “neutralization step” on themembrane fraction matrix to yield the Membrane-Derived CosmeticComposition of the present invention.

In one embodiment, the stabilization step involves mixing a non-ionicemulsifier and at least one antioxidant with the membrane fraction toyield a stabilized membrane fraction component. In the polymer matrixincorporation step, the stabilized membrane fraction component isincorporated into a polymer matrix to yield a membrane fraction matrix.Suitable polymers for use in the present invention include, for example,at least one polymeric emulsifier and at least one preservative. Themembrane fraction matrix is then subjected to the neutralization step,which step involves adjusting the pH of the membrane fraction matrix toa range of between 2.5 and 6.5, yielding the Membrane-Derived CosmeticComposition described in the present application.

In another embodiment, stabilization of the Membrane-Derived CosmeticComposition to yield approximately 100 grams of the composition isperformed as follows:

(a) Stabilization of Membrane Fraction Includes its Mixing withNon-Ionic Emulsifier Polysorbate 80 (Tween 80) and Antioxidants (Tenox4).

As an example, 20 grams fresh membrane fraction are mixed vigorouslyuntil homogeneous with 3.5 grams of Tween 80 and 0.1 gram of Tenox 4(solution of Butylated Hydroxyanisole and Butylated Hydroxytoluene inoil) while avoiding aeration during mixing.

(b) Preparation of the Dispersion of Polymeric Emulsifier,Acrylates/C10-C30 Acrylate Crosspolymer:

As an example, 0.9 gram Pemulen TR-2 was dispersed in 69.2 grams warmdeionized water and mixed until uniform using moderate agitation,avoiding aeration. In parallel, 5 grams of Glycerin and 1 gram ofPhenonip (mixture of Phenoxyethanol (and) Methylparaben (and)Butylparaben (and) Ethylparaben (and) Propylparaben) are combined inseparate vessel and mixed until uniform. With moderate agitation, phasescontaining Pemulen and Glycerin with Phenonip are combined and mixeduntil uniform.

(c) Incorporation of Membrane Fraction into Polymer Matrix:

As an example, the phase containing membrane fraction, Tween 80 andTenox 4 is added to the phase containing Pemulen, Glycerin and Phenonipand mixed with vigorous agitation while avoiding aeration.

(d) Neutralization of the Product:

As an example, the batch containing membrane fraction and othercomponents is neutralized with 18% aqueous solution of Sodium Hydroxide(NaOH) and mixed vigorously to produce uniform system having pH=5.0±0.4.

The resulting multiphase products are opaque gels demonstratingproperties that fully satisfy all requirements to the cosmeticingredients. It was found that optimum composition of preservatives andanti-oxidants in multiphase cosmetic ingredients is very similar for allplant sources and different combinations of Pemulens and also Carbopolscan be effectively used. Stability studies indicate that cosmeticingredients produced from membrane fractions via methods described arestable at 8° C. for at least 4-6 months maintaining physico-chemicalintegrity and activities.

Process for Preparing Serum-Derived Cosmetic Compositions

The present invention also relates to a method for preparing theSerum-Derived Cosmetic Composition exhibiting antioxidant activity, cellgrowth stimulation activity, or both antioxidant and cell growthstimulation activities. The method involves providing a plant cell juicethat has been extracted from a fresh plant biomass, as already describedabove with respect to the Membrane-Derived Cosmetic Composition. Theplant cell juice is then treated under conditions effective to separatethe plant cell juice into a membrane fraction and a cell juicesupernatant. This step is also the same as described above with respectto the Membrane-Derived Cosmetic Composition. The cell juice supernatantis processed under conditions effective to separate the cell juicesupernatant into a cytoplasm fraction and a cell serum fraction. Thecell serum fraction is refined under conditions effective to yield acell serum fraction filtrate having antioxidant activity, cell growthstimulation activity, or both antioxidant and cell growth stimulationactivities. The cell serum fraction filtrate is stabilized underconditions effective to yield a stable bioactive botanical cosmeticcomposition exhibiting antioxidant activity, cell growth stimulationactivity, or both antioxidant and cell growth stimulation activities.

The plant cell juice may be extracted from all types of plants. Examplesof suitable plants that may be used as sources of fresh plant biomass inthe present include, without limitation, plants from the followingfamilies: Asteraceae, Fabaceae, Lamiaceae, and Poaceae. In particular,examples of specific plants that have been tested and found appropriateas fresh plant biomass sources include, without limitation, Trifoliumpratense, Nelumbo nucifera, Calendula officinalis, Medicago sativa,Lavandula angustifolia, Salvia officinalis, and Hordeum vulgare.

As described above, once the plant cell juice is separated into amembrane fraction and a cell juice supernatant, the cell juicesupernatant is subjected to a processing step. In one embodiment, theprocessing step involves (1) performing a “cytoplasmic fractionprecipitation step” to yield a cytoplasm/cell serum mixture includingthe cytoplasmic fraction and the cell serum fraction, and (2) performinga “cell serum separation step” to separate the cytoplasmic fraction fromthe cell serum fraction. The cytoplasmic fraction includes predominantlywhite soluble proteins; in C3 plants, these proteins largely consist ofthe enzyme ribulose biphosphate carboxilase. The cell serum contains lowmolecular weight soluble components.

The cytoplasmic fraction precipitation step may include inducingprecipitation of the cytoplasmic fraction within the cell juicesupernatant using a suitable precipitation technique, including, forexample, isoelectric titration and electrodialysis. In one embodiment,the isoelectric titration involves adjusting the pH of the cell juicesupernatant to between about 2.5 and 6.5. The cytoplasm/cell serummixture is induced to separate into a cytoplasmic fraction and a cellserum fraction using a suitable separation technique, including, forexample, such techniques as filtration and centrifugation. As anexample, the precipitation was induced by a titration method utilizingby 5.0N Hydrochloric Acid (HCl) to pH=4.0.

The quantitative criteria to evaluate the complete separation ofcytoplasm fraction is the absence of detectable levels of high molecularweight proteins and/or the absence of ribulose biphosphate carboxilasein subsequent filtrate or supernatant. As an example, the precipitatedcell juice supernatants may be separated in a refrigerated centrifugefor greater than or equal to 20 minutes at greater than or equal to3,000 g, and an absence of the proteins having molecular weight ofgreater than or equal to 10,000 in cell serum having pH=4.0 wereachieved.

The cell serum is commonly referred to as “brown juice,” althoughinitially this clear liquid has a slight yellow color and slightcharacteristic odor. In several hours, the unstable cell serum isirreversibly transformed into dark brown color suspension containingheavy precipitate and strong non-characteristic odor. As a result,“brown juice” cannot be used as a cosmetic ingredient. The describedprocedure that follows allows for the refinement of cell serum (brownjuice) to yield a stable and active cosmetic ingredients. This isaccomplished by removing from the cell serum the major componentsresponsible for the irreversible transformations that lead to generationof unwanted precipitate and deterioration of color and odor. Thisprocedure includes: pH adjustment, heat treatment, cooling, vacuumfiltration, and stabilization. Some specific regiment procedures mayvary according to plant source cell serum. It should be noted that thisprocedure must be used immediately after separation of cell serum fromcytoplasm fraction is completed.

Once the cell serum fraction is produced, it is the subjected to arefining process. This refining process includes (1) performing a“temperature treatment step” to yield a coagulated cell serum fraction,and (2) performing a clarification step to yield a cell serum fractionfiltrate. A suitable temperature treatment step for use in the presentinvention involves (1) heating the cell serum fraction to a heatingtemperature required to induce coagulation within the cell serumfraction, and (2) immediately cooling the cell serum fraction to atemperature effective to allow further quantitative separation of saidcell serum fraction filtrate. In one embodiment, the heating temperatureis between about 80 and about 95 degrees Celsius, and the cooling of theheated cell serum fraction is to a temperature of at least as low asabout 15 degrees Celsius. A suitable clarification step for use in thepresent invention involves clarifying the coagulated cell serum fractionto yield a cell serum fraction filtrate, where the clarifying involvesclarification techniques such as filtration and centrifugation. In oneembodiment, the filtration may involve vacuum filtrating the coagulatedcell serum fraction to yield the cell serum fraction filtrate. Inanother embodiment, prior to the temperature treatment step, the cellserum fraction is adjusted to a pH of between about 3.0 and 4.0, asappropriate.

After the cell serum filtrate is produced, it is then subjected to thestabilizing step mentioned above to yield the Serum-Derived CosmeticComposition. In one embodiment, the stabilizing step involves incubatingthe cell serum fraction filtrate in a mixture of at least onepreservative and at least one antioxidant to yield a stabilized cellserum fraction filtrate. Suitable preservatives for use in the presentinvention include, for example, potassium sorbate, sodium benzoate,sodium methyl paraben, and citric acid. An example of a suitableantioxidant for use in the present invention is sodium metabisulfite.

In one embodiment, stabilization of the cell serum may be performed asfollows:

(a) As an example, the pH adjustment is performed for cell serumobtained from sage and marigold flowers induced by a titration methodutilizing by 5.0N Hydrochloric Acid (HCl) to pH=3.0. Such adjustment isnot necessary for cell serum obtained from alfalfa, barley and lavender.

(b) Heat treatment is performed for cell sera obtained from all useableplant sources. As an example, cell sera are exposed to microwavetreatment under the temperature probe control. This treatment iscontinued until the temperature reaches 90° C. The temperature probeindicates the point required to induce the complete coagulation of theunwanted components. Once coagulation is induced, the treated cell juiceis immediately cooled to 10° C.

(c) The coagulated cell sera can be clarified by filtration orcentrifugation. As an example, the coagulated cell sera may be vacuumfiltrated through double layers of Whatman No. 2 filters. Theprecipitates are discarded, and filtrates are used for furtherprocessing.

(d) Stabilization of the filtrates included addition of specificpreservatives and anti-oxidants and incubation of the mixtures untiltheir complete solubilization is achieved (usually greater than or equalto 30 minutes of extensive mixing is required).

Stabilized cell serum filtrates demonstrate properties which fullysatisfy all requirements of cosmetic ingredients. Stability studiesindicate that cosmetic ingredients produced from cell serum via thesemethods are stable at room temperature for at least 10-12 months (i.e.,they maintain physico-chemical integrity and activities).

EXAMPLES Example 1 Preparation of Cosmetic Botanical Ingredient 101Derived from Alfalfa (Medicago sativa) Cell Serum Fractions

Biomass Preparation. Sufficient amounts of fresh alfalfa (Medicagosativa) plant biomass (i.e., stem and leaf tissue) were harvested toyield approximately 100 kg of dry matter. The level of dry matter in thefresh alfalfa plant biomass was calculated to be 15.75 percent,requiring harvesting of approximately 635 kg of fresh alfalfa plantbiomass to yield 100 kg of dry matter. Care was taken to preserve theinherent moisture content of the plant biomass and to avoid wilting dueto moisture loss. The plants were cut at least 5 centimeters above theground to limit the amount of soil and other debris in the collectedplant biomass. The cutting was conducted in such a manner as to avoid orminimize chopping, mashing, and crushing of the plants. The harvestedplants were delivered for processing not more than 60 minutes aftercutting. This was done to minimize exposure of the plant biomass to sun,high temperature, and other negative environmental factors. A washingstep was performed to remove soil particles and other debris from theplants prior to further processing. This washing was accomplished bywashing the harvested plants for ≦5 minutes in ≦1 kg/cm² water pressure.The residual water wash did not contain any green pigments, indicatingproper water pressure and washing duration. The excess water was removedfrom the washed plant biomass.

Grinding, Maceration, and Pressing of Plant Biomass.

After harvesting, collecting, and washing the plant biomass, the plantsthen underwent grinding, maceration, and pressing to extract theintracellular content (i.e., the plant cell juice) and to separate itfrom the fiber-enriched press-cake. A hammer mill was used to grind thealfalfa biomass to yield plant tissue particles of suitably small sizein a short amount of time and without significant increase of biomasstemperature. The hammer mill was set to produce the maximum size ofmacerated plant particles of ≦0.5 centimeters during ≦10 seconds oftreatment. This resulted in only an increase of ≦5° C. biomasstemperature. A horizontal continuous screw press (Compact Press “CP-6”,Vincent Corporation, FL) was used to extract the plant cell juice fromthe plant biomass. The pressure on the cone of the screw press wasmaintained at a level of 24 kg/cm², with a screw speed of 12 rpm andonly a temperature increase of ≦5° C. This treatment yielded thepress-cake and the plant cell juice. The initial plant cell juicecontained small fiber particles, which were removed by filtrationthrough four layers of nylon fabric or by using low-speedcentrifugation.

Separation of the Membrane Fraction from the Cell Juice.

The filtered plant cell juice was exposed to microwave treatment using atemperature probe control. This treatment continued until thetemperature of the cell juice reached 60° C. Once coagulation wasinduced, the treated cell juice was immediately cooled to 40° C.Separation of the membrane fraction from the coagulated cell juice wasachieved using centrifugation at greater than or equal to 3,000 g forgreater than or equal to 20 minutes. This yielded a membrane fraction(precipitate) and a cell juice supernatant, which contained a cytoplasmfraction and a cell serum fraction (i.e., low molecular weight solublecomponents). The cell juice supernatant was used for further processingto yield Cosmetic Botanical Ingredient 101. The membrane fraction waspreserved for use in preparing a counterpart Membrane-Derived CosmeticComposition.

Separation of the Cytoplasm Fraction from the Cell Juice Supernatant.

In order to separate out the cytoplasm fraction, the cell juicesupernatant was subjected to isoelectric precipitation. Precipitation ofthe cytoplasm fraction was induced using a titration method utilizing5.0 N hydrochloric acid (HCl) to bring the pH of the cell juicesupernatant to 4.0. The separation of precipitated cytoplasm fractionfrom the cell serum was achieved by centrifugation at greater than orequal to 3,000 g for greater than or equal to 20 minutes. This resultedin a cell serum (supernatant) that could be further refined to yieldBotanical Cosmetic Ingredient 101.

Treatment of the Cell Serum to Produce Cosmetic Botanical Ingredient101.

The refinement of the cell serum involved the following steps: heattreatment, cooling, filtration, and stabilization. Refinement wasperformed immediately after separation of the cell serum from thecytoplasm fraction. The cell serum was exposed to microwave treatmentusing a temperature probe control. This treatment continued until thetemperature of the cell serum reached 90° C. Once coagulation wasinduced, the treated cell serum was immediately cooled to 10° C. Thecoagulated cell serum was vacuum filtrated through double layers ofWhatman No. 2 filters. The precipitate was discarded and the resultingcell serum filtrate was used for further processing (i.e.,stabilization). Stabilization of the cell serum filtrate was achieved byadding preservatives and antioxidants and incubating the mixture untilcomplete solubilization was achieved. The preservatives and antioxidantsused included the following: 0.1% potassium sorbate, 0.1% sodiumbenzoate, 0.1% sodium methyl paraben, and 0.2% sodium metabisulfite.This preparation resulted in the production of 18.1 kg of Dry Matteryield (or approximately 340 Liters) of the Cosmetic Botanical Ingredient101, which was used for characterization of its physico-chemical andbioactive qualities. The recommended storage conditions for CosmeticBotanical Ingredient 101 include storage in a closed container protectedfrom light at a temperature of between 15 and 25° C.

Example 2 Product Specifications of Cosmetic Botanical Ingredient 101Derived from Alfalfa (Medicago sativa) Cell Serum Fractions

Cosmetic Botanical Ingredient 101 was prepared according to the processdescribed above in Example 1. Analyses of Cosmetic Botanical Ingredient101 were conducted to determine its various physico-chemical, microbial,cytotoxicity, and bioactivity characteristics, as described below.Cosmetic Botanical Ingredient 101 is a clear liquid, which has alight-yellow color and a light-characteristic odor. No solvent (i.e.glycol, oil, or water) was added to the carrier medium.

Table 1 summarizes the Physical and Chemical data of Cosmetic BotanicalIngredient 101.

TABLE 1 Physical and Chemical Data Parameter Method Results SolidContent, % See Example 25, 5.3 “Method 1” Specific Gravity, g/cm³ USP<841> 1.025 Color Gardner Scale 6-7 Refractive Index USP <831> 1.342 pHUSP <791> 4.1 Red-Ox Potential, mV See reference [1] 70 Conductivity,S/m See reference [2] 0.96 Reference: [1] Handbook of Chemistry andPhysics, 80^(th) Edition, CRC Press, 1999-2000, 5-90; [2] Handbook ofChemistry and Physics, 80^(th) Edition, CRC Press, 1999-2000, 8-21,which are hereby incorporated by reference in their entirety.

Table 2 describes the UV-Spectra data regarding Cosmetic BotanicalIngredient 101.

TABLE 2 UV-Spectra Peak Parameter Method Results #1 Start, nm USP <197>400.0 Apex, nm ″ 324.5 End, nm ″ 303.0 Height, Abs ″ 0.347 Area, Abs ×nm ″ 21.197 #2 Start, nm USP <197> 303.0 Apex, nm ″ 258.0 End, nm ″233.0 Height, Abs ″ 1.471 Area, Abs × nm ″ 65.103

Table 3 summarizes the microbial analysis data for Cosmetic BotanicalIngredient 101. This data demonstrates that Cosmetic BotanicalIngredient 101 satisfies the cosmetic industry requirements regardingcolony forming units and absence of pathogens.

TABLE 3 Microbial Analysis Parameter Method Results Colony Forming UnitsUSP <61> <100 (CFU) per gram of sample Escherichia coli ″ NegativeCandida albicans ″ Negative Pseudomonas sp. ″ Negative Staphylococcusaureus ″ Negative

Cosmetic Botanical Ingredient 101 was determined to be stable (i.e.,maintaining physical and chemical integrity) for at least 12-18 monthswhile stored at a temperature of between 15 and 25° C. in a closedcontainer protected from light. No toxic effect was detected. In acontrolled clinical evaluation, Cosmetic Botanical Ingredient 101 didnot demonstrate 50% inhibition of neutral red uptake (NRU₅₀) by 3T3fibroblasts in the concentration range 0-2,500 μg dry matter/ml. TheNRU₅₀ of positive control (epidermal growth factor)>2,500 μg/ml.Cosmetic Botanical Ingredient 101 demonstrated superoxide scavengingability. In a controlled clinical evaluation, Cosmetic BotanicalIngredient 101 demonstrated a 50% inhibition of cytochrome c reduction(ICR₅₀) at a concentration 149 μg dry matter/ml. The ICR₅₀ of positivecontrol (rosmarinic acid)=26.5 μg/ml. Cosmetic Botanical Ingredient 101is a biodegradable product.

Example 3 Preparation of Cosmetic Botanical Ingredient 201 Derived fromBarley (Hordeum vulgare) Cell Serum Fractions

The process for preparing Cosmetic Botanical Ingredient 201 wasidentical to the process described in Example 1 with regard to CosmeticBotanical Ingredient 101, with the variations noted below. Fresh stemand leaf tissue of barley (Hordeum vulgare) was used as the plantbiomass starting material. The level of dry matter in the fresh barleyplant biomass was calculated to be 13.67 percent, requiring harvestingof approximately 732 kg of fresh barley plant biomass to yield 100 kg ofdry matter. The preparation resulted in the production of 15.1 kg of DryMatter yield (or approximately 433 liters) of Cosmetic BotanicalIngredient 201.

Example 4 Product Specifications of Cosmetic Botanical Ingredient 201Derived from Barley (Hordeum vulgare) Cell Serum Fractions

Cosmetic Botanical Ingredient 201 was prepared according to the processdescribed above in Example 3. Analyses of Cosmetic Botanical Ingredient201 were conducted to determine its various physico-chemical, microbial,cytotoxicity, and bioactivity characteristics, as described below.Cosmetic Botanical Ingredient 201 is a clear liquid, which has alight-yellow color and a light-characteristic odor. No solvent (i.e.,glycol, oil, or water) was added to the carrier medium.

Table 4 summarizes the Physical and Chemical data of Cosmetic BotanicalIngredient 201.

TABLE 4 Physical and Chemical Data Parameter Method Results SolidContent, % See Example 25, 3.5 “Method 1” Specific Gravity, g/cm³ USP<841> 1.019 Color Gardner Scale 5-6 Refractive Index USP <831> 1.338 pHUSP <791> 4.1 Red-Ox Potential, mV See reference [1] 95 Conductivity,S/m See reference [2] 1.09 Reference: [1] Handbook of Chemistry andPhysics, 80^(th) Edition, CRC Press, 1999-2000, 5-90; [2] Handbook ofChemistry and Physics, 80^(th) Edition, CRC Press, 1999-2000, 8-21,which are hereby incorporated by reference in their entirety.

Table 5 summarizes the UV-Spectra data for Cosmetic Botanical Ingredient201.

TABLE 5 UV-Spectra Peak Parameter Method Results #1 Start, nm USP <197>400.0 Apex, nm ″ 333.5 End, nm ″ 305.0 Height, Abs ″ 0.232 Area, Abs ×nm ″ 14.254 #2 Start, nm USP <197> 305.0 Apex, nm ″ 258.0 End, nm ″233.0 Height, Abs ″ 1.268 Area, Abs × nm ″ 55.631

Microbial analyses demonstrated that Cosmetic Botanical Ingredient 201satisfies the cosmetic industry requirements for cosmetic ingredientswith regard to CFUs and absence of pathogens (see Table 3, above, formethods).

Cosmetic Botanical Ingredient 201 was determined to be stable (i.e.,maintaining physical and chemical integrity) for at least 12-18 monthswhile stored at a temperature of between 15 and 25° C. in a closedcontainer protected from light. No toxic effect was detected. In acontrolled clinical evaluation, Cosmetic Botanical Ingredient 201 didnot demonstrate 50% inhibition of neutral red uptake (NRU₅₀) by 3T3fibroblasts in the concentration range 0-2,500 μg dry matter/ml. TheNRU₅₀ of positive control (epidermal growth factor)>2,500 μg/ml.Cosmetic Botanical Ingredient 201 demonstrated superoxide scavengingability. In a controlled clinical evaluation, Cosmetic BotanicalIngredient 201 demonstrated a 50% inhibition of cytochrome c reduction(ICR₅₀) at a concentration 160 μg dry matter/ml. The ICR₅₀ of positivecontrol (rosmarinic acid)=2.65 μg/ml. Cosmetic Botanical Ingredient 201is a biodegradable product.

Example 5 Preparation of Cosmetic Botanical Ingredient 301 Derived fromLavender (Lavandula angustifolia) Cell Serum Fractions

The process for preparing Cosmetic Botanical Ingredient 301 wasidentical to the process described in Example 1 with regard to CosmeticBotanical Ingredient 101, with the variations noted below. Fresh stemand leaf tissue of lavender (Lavandula angustifolia) was used as theplant biomass starting material. The level of dry matter in the freshlavender plant biomass was calculated to be 13.24 percent, requiringharvesting of approximately 755 kg of fresh lavender plant biomass toyield 100 kg of dry matter. Also, the preservative and antioxidantmixture contained the following: 0.1% potassium sorbate, 0.1% sodiumbenzoate, 0.1% sodium methyl paraben, 0.1% citric acid, and 0.2% sodiummetabisulfite. The preparation resulted in the production of 18.5 kg ofDry Matter yield (or approximately 444 liters) of Cosmetic BotanicalIngredient 301.

Example 6 Product Specifications of Cosmetic Botanical Ingredient 301Derived from Lavender (Lavandula angustifolia) Cell Serum Fractions

Cosmetic Botanical Ingredient 301 was prepared according to the processdescribed above in Example 5. Analyses of Cosmetic Botanical Ingredient301 were conducted to determine its various physico-chemical, microbial,cytotoxicity, and bioactivity characteristics, as described below.Cosmetic Botanical Ingredient 301 is a clear liquid, which has abrown-yellow color and a characteristic odor. No solvent (i.e., glycol,oil, or water) was added to the carrier medium.

Table 6 summarizes the Physical and Chemical data of Cosmetic BotanicalIngredient 301.

TABLE 6 Physical and Chemical Data Parameter Method Results SolidContent, % See Example 25, 4.2 “Method 1” Specific Gravity, g/cm³ USP<841> 1.020 Color Gardner Scale 11-12 Refractive Index USP <831> 1.341pH USP <791> 3.9 Red-Ox Potential, mV See reference [1] 170Conductivity, S/m See reference [2] 0.79 Reference: [1] Handbook ofChemistry and Physics, 80^(th) Edition, CRC Press, 1999-2000, 5-90; [2]Handbook of Chemistry and Physics, 80^(th) Edition, CRC Press,1999-2000, 8-21, which are hereby incorporated by reference in theirentirety.

Table 7 describes the UV-Spectra data regarding Cosmetic BotanicalIngredient 301.

TABLE 7 UV Spectra Peak Parameter Method Results #1 Start, nm USP <197>400.0 Apex, nm ″ 260.5 End, nm ″ 236.5 Height, Abs ″ 2.409 Area, Abs ×nm ″ 135.505

Microbial analyses demonstrated that Cosmetic Botanical Ingredient 301satisfies the cosmetic industry requirements for cosmetic ingredientswith regard to CFUs and absence of pathogens (see Table 3, above, formethods).

Cosmetic Botanical Ingredient 301 was determined to be stable (i.e.,maintaining physical and chemical integrity) for at least 12-18 monthswhile stored at a temperature of between 15 and 25° C. in a closedcontainer protected from light. No toxic effect was detected. In acontrolled clinical evaluation, Cosmetic Botanical Ingredient 301 didnot demonstrate 50% inhibition of neutral red uptake (NRU₅₀) by 3T3fibroblasts in the concentration range 0-400 μg dry matter/ml. The NRU₅₀of positive control (epidermal growth factor)>2,500 μg/ml. CosmeticBotanical Ingredient 301 demonstrated elastase inhibitory activity,gelatinase B inhibitory activity, and superoxide scavenging ability.

Table 8, below, describes the bioactivity results regarding CosmeticBotanical Ingredient 301.

TABLE 8 Bioactivity Results of Cosmetic Botanical Ingredient 301 IC₅₀K_(i) Activity Method (μg/ml) (μg/ml) Elastase Inhibitory See Example25, 36.0 25.4 “Method 5” Gelantinase B inhibitory See Example 25, >100No data “Method 6”

In a controlled clinical evaluation, Cosmetic Botanical Ingredient 301demonstrated a 50% inhibition of cytochrome c reduction (ICR₅₀) at aconcentration 158 μg dry matter/ml. The ICR₅₀ of positive control(rosmarinic acid)=26.5 μg/ml. Cosmetic Botanical Ingredient 301 is abiodegradable product.

Example 7 Preparation of Cosmetic Botanical Ingredient 401 Derived fromMarigold Flower (Calendula officinalis) Cell Serum Fractions

The process for preparing Cosmetic Botanical Ingredient 401 wasidentical to the process described in Example 1 with regard to CosmeticBotanical Ingredient 101, with the variations noted below. Fresh flowertissue of marigold (Calendula officinalis) was used as the plant biomassstarting material. The level of dry matter in the fresh marigold flowerplant biomass was calculated to be 7.80 percent, requiring harvesting ofapproximately 1,282 kg of fresh marigold flower plant biomass to yield100 kg of dry matter. The flowers were separated from the whole plantsafter cutting the plant and prior to washing. The processing of theflowers (i.e., beginning with the washing step and prior to grinding)started not more than 3 to 4 hours after cutting of the plant. Also,prior to microwave treatment of the cell serum fraction, the pH of thecell serum was first adjusted to a pH of 3.0, using a titration methodutilizing 0.5 N hydrochloric acid (HCl). The preparation resulted in theproduction of 27.1 kg of Dry Matter yield (or approximately 704 liters)of Cosmetic Botanical Ingredient 401.

Example 8 Product Specifications of Cosmetic Botanical Ingredient 401Derived from Marigold Flower (Calendula officinalis) Cell SerumFractions

Cosmetic Botanical Ingredient 401 was prepared according to the processdescribed above in Example 7. Analyses of Cosmetic Botanical Ingredient401 were conducted to determine its various physico-chemical, microbial,cytotoxicity, and bioactivity characteristics, as described below.Cosmetic Botanical Ingredient 401 is a clear liquid, which has alight-yellow color and a light-characteristic color. No solvent (i.e.,glycol, oil, or water) has been added to the carrier medium.

Table 9 describes the Physical and Chemical data of Cosmetic BotanicalIngredient 401.

TABLE 9 Physical and Chemical Data Parameter Method Results SolidContent, % See Example 25, 3.9 “Method 1” Specific Gravity, g/cm³ USP<841> 1.019 Color Gardner Scale 4-5 Refractive Index USP <831> 1.340 pHUSP <791> 3.4 Red-Ox Potential, mV See reference [1] 160 Conductivity,S/m See reference [2] 0.40 References: [1] Handbook of Chemistry andPhysics, 80^(th) Edition, CRC Press, 1999-2000, 5-90; [2] Handbook ofChemistry and Physics, 80^(th) Edition, CRC Press, 1999-2000, 8-21,which are hereby incorporated by reference in their entirety.

Table 10 summarizes the UV-Spectra data for Cosmetic BotanicalIngredient 401.

TABLE 10 UV-Spectra Peak Parameter Method Results #1 Start, nm USP <197>400.0 Apex, nm ″ 260.0 End, nm ″ 232.5 Height, Abs ″ 1.171 Area, Abs ×nm ″ 55.719

Microbial analyses demonstrated that Cosmetic Botanical Ingredient 401satisfies the cosmetic industry requirements for cosmetic ingredientswith regard to CFUs and absence of pathogens (see Table 3, above, formethods).

Cosmetic Botanical Ingredient 401 was determined to be stable (i.e.,maintaining physical and chemical integrity) for at least 12-18 monthswhile stored at a temperature of between 15 and 25° C. in a closedcontainer protected from light. No toxic effect was detected. In acontrolled clinical evaluation, Cosmetic Botanical Ingredient 401 didnot demonstrate 50% inhibition of neutral red uptake (NRU₅₀) by 3T3fibroblasts in the concentration range 0-2,500 μg dry matter/ml. TheNRU₅₀ of positive control (epidermal growth factor)>2,500 μg/ml.Cosmetic Botanical Ingredient 401 demonstrated stimulation effect oncell proliferation and superoxide scavenging ability. In a controlledclinical evaluation, Cosmetic Botanical Ingredient 401 stimulated 3T3fibroblasts proliferation. This 10-15% stimulation was observed over arange from 5 to 100 μg dry matter/ml. The stimulation by positivecontrol (epidermal growth factor)=20-30%. In a controlled clinicalevaluation, Cosmetic Botanical Ingredient 401 demonstrated superoxidescavenging activity resulting in 50% inhibition of cytochrome creduction (ICR₅₀) at a concentration 153 μg dry matter/ml. The ICR₅₀ ofpositive control (rosmarinic acid)=26.5 μg/ml. Cosmetic BotanicalIngredient 401 is a biodegradable product.

Example 9 Preparation of Cosmetic Botanical Ingredient 501 Derived fromSage (Salvia officinalis) Cell Serum Fractions

The process for preparing Cosmetic Botanical Ingredient 501 wasidentical to the process described in Example 1 with regard to CosmeticBotanical Ingredient 101, with the variations noted below. Fresh stemand leaf tissue of sage (Salvia officinalis) was used as the plantbiomass starting material. The level of dry matter in the fresh sageplant biomass was calculated to be 10.64 percent, requiring harvestingof approximately 940 kg of fresh sage plant biomass to yield 100 kg ofdry matter. Prior to microwave treatment of the cell serum fraction, thepH of the cell serum was first adjusted to a pH of 3.0, using atitration method with 0.5 N hydrochloric acid (HCl). Also, thepreservative and antioxidant mixture contained the following: 0.1%potassium sorbate, 0.1% sodium benzoate, 0.1% sodium methyl paraben,0.1% citric acid, and 0.2% sodium metabisulfite. The preparationresulted in the production of 14.9 kg of Dry Matter yield (orapproximately 370 liters) of Cosmetic Botanical Ingredient 501.

Example 10 Product Specifications of Cosmetic Botanical Ingredient 501Derived from Sage (Salvia officinalis) Cell Serum Fractions

Cosmetic Botanical Ingredient 501 was prepared according to the processdescribed above in Example 9. Analyses of Cosmetic Botanical Ingredient501 were conducted to determine its various physico-chemical, microbial,cytotoxicity, and bioactivity characteristics, as described below.Cosmetic Botanical Ingredient 501 is a clear liquid, which has abrown-yellow color and a characteristic odor. No solvent (i.e., glycol,oil, or water) was added to the carrier medium.

Table 11 describes the Physical and Chemical data of Cosmetic BotanicalIngredient 501.

TABLE 11 Physical and Chemical Data Parameter Method Results SolidContent, % See Example 25, 4.0 “Method 1” Specific Gravity, g/cm³ USP<841> 1.021 Color Gardner Scale 8-9 Refractive Index USP <831> 1.340 pHUSP <791> 3.2 Red-Ox Potential, mV See reference [1] 190 Conductivity,S/m See reference [2] 0.99 References: [1] Handbook of Chemistry andPhysics, 80^(th) Edition, CRC Press, 1999-2000, 5-90; [2] Handbook ofChemistry and Physics, 80^(th) Edition, CRC Press, 1999-2000, 8-21,which are hereby incorporated by reference in their entirety.

Table 12 summarizes the UV-Spectra data for Cosmetic BotanicalIngredient 501.

TABLE 12 UV-Spectra Peak Parameter Method Results #1 Start, nm USP <197>400.0 Apex, nm ″ 330.0 End, nm ″ 306.5 Height, Abs ″ 0.260 Area, Abs ×nm ″ 14.952 #2 Start, nm USP <197> 306.5 Apex, nm ″ 259.0 End, nm ″235.0 Height, Abs ″ 1.248 Area, Abs × nm ″ 57.844

Microbial analyses demonstrated that Cosmetic Botanical Ingredient 501satisfies the cosmetic industry requirements for cosmetic ingredientswith regard to CFUs and absence of pathogens (see Table 3, above, formethods).

Cosmetic Botanical Ingredient 501 was determined to be stable (i.e.,maintaining physical and chemical integrity) for at least 12-18 monthswhile stored at a temperature of between 15 and 25° C. in a closedcontainer protected from light. No toxic effect was detected. In acontrolled clinical evaluation, Cosmetic Botanical Ingredient 501 didnot demonstrate 50% of neutral red uptake (NRU₅₀) by 3T3 fibroblasts inthe concentration range 0-2,430 μg dry matter/ml. The NRU₅₀ of positivecontrol (epidermal growth factor)>2,500 μg/ml. Cosmetic BotanicalIngredient 501 demonstrated elastase inhibitory activity, gelatinase Binhibitory activity, and superoxide scavenging ability. (See Table 13,below.)

TABLE 13 Bioactivity Results of Cosmetic Botanical Ingredient 501 IC₅₀K_(i) Activity Method (μg/ml) (μg/ml) Elastase Inhibitory See Example25, 115.0 70.3 “Method 5” Gelatinase B Inhibitory See Example 25, >100No data “Method 6”

In a controlled clinical evaluation, Cosmetic Botanical Ingredient 501demonstrated superoxide scavenging activity, resulting in 50% inhibitionof cytochrome c reduction (ICR₅₀) at a concentration >160 μg drymatter/ml. The ICR₅₀ of positive control (rosmarinic acid)=26.5 μg/ml.Cosmetic Botanical Ingredient 501 is a biodegradable product.

Example 11 Preparation of Cosmetic Botanical Ingredient 402 Derived fromMarigold Flower (Calendula officinalis) Membrane Fractions

The process for preparing Cosmetic Botanical Ingredient 402 wasidentical to the process described in Example 7 with regard to CosmeticBotanical Ingredient 401, with the variations noted below. Once themembrane fraction (precipitate) was separated from the filtered celljuice, the process described in Example 7 was no longer followed.Instead, the membrane fraction was treated to yield Cosmetic BotanicalIngredient 402, as described below.

Treatment of Membrane Fraction to Produce Cosmetic Botanical Ingredient402.

The membrane fraction was stabilized and incorporated into a polymermatrix. This was performed immediately after separation of the membranefraction from cell juice. To prepare approximately 100 grams of CosmeticBotanical Ingredient 402, the cell membrane fraction was stabilized bymixing it with non-ionic emulsifier Polysorbate 80 (Tween 80) andantioxidants (Tenox 4). Specifically, 20 grams of fresh membranefraction was mixed vigorously with 3.5 grams of Tween 80 and 0.1 gram ofTenox 4 (solution of Butylated Hydroxyanisole and ButylatedHydroxytoluene in oil) until homogeneous, while avoiding aeration duringmixing.

Once stabilized, the membrane fraction was incorporated into a polymermatrix (i.e., a dispersion of polymeric emulsifier, acrylates/C10-C30acrylate crosspolymer). The polymer matrix was prepared by dispersing0.9 grams of Pemulen TR-2 in 69.2 grams of warm deionized water andmixing until uniform using moderate agitation, while avoiding aeration.In parallel, 5 grams of Glycerin and 1.0 gram of Phenonip (mixture ofPhenoxyethanol (and) Methylparaben (and) Butylparaben (and) Ethylparaben(and) Propylparaben) were combined in a separate vessel and mixed untiluniform. With moderate agitation, the phases containing Pemulen andGlycerin with Phenonip were combined and mixed until uniform. Toincorporate the membrane fraction into the polymer matrix, the phasecontaining the membrane fraction, Tween 80, and Tenox 4 was added to thephase containing the Pemulen, Glycerin, and Phenonip, and then mixedwith vigorous agitation while avoiding aeration. Stabilization of themembrane fraction mixture was achieved by neutralizing it with 18%aqueous solution of sodium hydroxide (NaOH) and mixed vigorously toproduce a uniform system having a pH of 5.0±0.4. This preparation, whichstarted from 100 kg of fresh marigold flower plant biomass(approximately 1,282 kg of fresh marigold flower biomass having 7.80percent dry matter), resulted in the production of 9.5 kg of Dry Matteryield (or approximately 205 liters) of Cosmetic Botanical Ingredient402, which was used for characterization of its physico-chemical andbioactive qualities. The recommended storage conditions for CosmeticBotanical Ingredient 402 include storage in a closed container protectedfrom light at a temperature between 2 and 8° C.

Example 12 Product Specifications of Cosmetic Botanical Ingredient 402Derived from Marigold Flower (Calendula officinalis) Membrane Fractions

Cosmetic Botanical Ingredient 402 was prepared according to the processdescribed above in Example 11. Analyses of Cosmetic Botanical Ingredient402 were conducted to determine its various physico-chemical, microbial,cytotoxicity, and bioactivity characteristics, as described below.Cosmetic Botanical Ingredient 402 is an opaque gel, which has an orangecolor and light-characteristic odor. Cosmetic Botanical Ingredient 402was formulated utilizing the natural cell juice constituents gelled witha polymer to assure the highest level of purity uniformity,compatibility, stability, safety and efficacy.

Table 14 describes the Physical and Chemical data of Cosmetic BotanicalIngredient 402.

TABLE 14 Physical and Chemical Data Parameter Method ResultsNon-Volatile Residue (NVR), % See Example 25, 7.1 “Method 2” SpecificGravity, g/cm³ USP <841> 1.054 Viscosity, cps USP <911> 15,800 pH USP<791> 4.6 Total Carotenoids, % NVR See Example 25, 0.86 “Method 4”Lutein, % NVR See Example 25, 0.83 “Method 4”

Table 15 summarizes the L*a*b* values data regarding Cosmetic BotanicalIngredient 402.

TABLE 15 L*a*b* Values Parameter Method Results L* See Example 25, 33.27“Method 3” a* See Example 25, 20.36 “Method 3” b* See Example 25, 49.56“Method 3”

Microbial analyses demonstrated that Cosmetic Botanical Ingredient 402satisfies the cosmetic industry requirements for cosmetic ingredientswith regard to CFUs and absence of pathogens (see Table 3, above, formethods).

Cosmetic Botanical Ingredient 402 was determined to be stable (i.e.,maintaining physical and chemical integrity) for at least 12-18 monthswhile stored at a temperature of between 2 and 8° C. in a closedcontainer protected from light. Cosmetic Botanical Ingredient 402 is abiodegradable product. No toxic effect was detected. In a controlledclinical evaluation, Cosmetic Botanical Ingredient 402 did notdemonstrate 50% inhibition of neutral red uptake (NRU₅₀) by 3T3fibroblasts in the concentration range 0-354 μg dry matter/ml. The NRU₅₀of positive control (epidermal growth factor)>2,500 μg/ml. CosmeticBotanical Ingredient 402 demonstrates elastase inhibitory activity andtrypsin inhibitory activity. Table 16 summarizes certain bioactivityresults for Cosmetic Botanical Ingredient 402.

TABLE 16 Bioactivity Results of Cosmetic Botanical Ingredient 402 IC₅₀K_(i) Activity Method (μg/ml) (μg/ml) Elastase Inhibitory See Example25, 21.0 0.68 “Method 5” Trypsin Inhibitory See reference [1] 5.6 Nodata Reference: [1] Carmel R. J. P., Kellam S. J., Owsianka A. M.,Walker J. M. Planta Medica, 1988, v. 54, pp. 10-14, which is herebyincorporated by reference in its entirety.

Example 13 Preparation of Cosmetic Botanical Ingredient 502 Derived fromSage (Salvia officinalis) Membrane Fractions

The process for preparing Cosmetic Botanical Ingredient 502 wasidentical to the process described in Example 11 with regard to CosmeticBotanical Ingredient 402, with the variations noted below. Fresh stemand leaf tissue of sage (Salvia officinalis) was used as the plantbiomass starting material. The level of dry matter in the fresh sageplant biomass was calculated to be 10.64 percent, requiring harvestingof approximately 940 kg of fresh sage plant biomass to yield 100 kg ofdry matter. This preparation resulted in the production of 6.7 kg of DryMatter yield (or approximately 124 liters) of Cosmetic BotanicalIngredient 502.

Example 14 Product Specifications of Cosmetic Botanical Ingredient 502Derived from Sage (Salvia officinalis) Membrane Fractions

Cosmetic Botanical Ingredient 502 was prepared according to the processdescribed above in Example 13. Analyses of Cosmetic Botanical Ingredient502 were conducted to determine its various physico-chemical, microbial,cytotoxicity, and bioactivity characteristics, as described below.Cosmetic Botanical Ingredient 502 is an opaque gel, which has a greencolor and characteristic odor. Cosmetic Botanical Ingredient 502 hasbeen formulated utilizing the natural cell juice constituents gelledwith a polymer to assure the highest level of purity uniformity,compatibility, stability, safety and efficacy.

Table 17 describes the Physical and Chemical data of Cosmetic BotanicalIngredient 502.

TABLE 17 Physical and Chemical Data Parameter Method ResultsNon-Volatile Residue, % See Example 25, 8.3 “Method 2” Specific Gravity,g/cm³ USP <841> 1.047 Viscosity, cps USP <911> 5.200 pH USP <791> 4.6

Table 18 describes the L*a*b* values for Cosmetic Botanical Ingredient502.

TABLE 18 L*a*b* Values Parameter Method Results L* See Example 25, 27.35“Method 3” a* See Example 25, −1.4 “Method 3” b* See Example 25, 16.97“Method 3”

Microbial analyses demonstrated that Cosmetic Botanical Ingredient 502satisfies the cosmetic industry requirements for cosmetic ingredientswith regard to CFUs and absence of pathogens (see Table 3, above, formethods).

Cosmetic Botanical Ingredient 502 was determined to be stable (i.e.,maintaining physical and chemical integrity) for at least 12-18 monthswhile stored at a temperature of between 2 and 8° C. in a closedcontainer protected from light. Cosmetic Botanical Ingredient 502 is abiodegradable product. No toxic effect was detected. Cosmetic BotanicalIngredient 502 demonstrates elastase inhibitory activity and gelatinaseB inhibitory activity. (See Table 19, below.)

TABLE 19 Bioactivity Results for Cosmetic Botanical Ingredient 502 IC₅₀K_(i) Activity Method (μg/ml) (μg/ml) Elastase Inhibitory See Example25, 30.0 12.0 “Method 5” Gelatinase B Inhibitory See Example 25, <25.0No data “Method 6”

Example 15 Distribution of Dry Matter Regarding Preparation of CosmeticBotanical Ingredients From Alfalfa, Barley, Lavender, Marigold Flowers,and Sage

Various fractions collected during the production of Cosmetic BotanicalIngredients 101, 201, 301, 401, 402, 501, and 502 were analyzed andcompared for dry matter distribution.

Table 20 shows the distribution of 100 kg dry mater between the celljuices and press-cakes of the various processes. It was determined thatthe process of the present invention permits extracted yield conversioninto plant cell juices in the range of from about 20 to 40 percent ofinitial biomass dry matter.

TABLE 20 Distribution of 100 kg Dry Matter Between Cell Juices andPress-Cakes Plant Source Marigold Product Alfalfa Barley LavenderFlowers Sage Fresh Biomass 100.0 100.0 100.0 100.0 100.0 Cell Juice 32.728.5 28.1 37.6 21.9 Press-Cake 67.3 71.5 71.9 62.4 78.1

Table 21 shows that the yield of membrane fractions' dry matter was inthe range from 6% to 13% of initial biomass dry matter and from 25% to45% of cell juice dry matter. Based on high dry matter yield, membranefractions were selected as a prospective source for preparation ofmultiphase cosmetic ingredients.

TABLE 21 Distribution of Dry Matter between Membrane Fractions and CellJuice Supernatants Plant Source Marigold Product Alfalfa Barley LavenderFlowers Sage Fresh Biomass 100.0 100.0 100.0 100.0 100.0 Cell Juice 32.728.5 28.1 37.6 21.9 Membrane Fraction 12.2 12.9 8.7 9.5 6.7 Cell JuiceSupernatant 20.5 15.6 19.4 28.1 15.2

The process of the present invention permitted the followingdistribution of dry matter between cytoplasm fractions and cell serum(see Table 22).

TABLE 22 Distribution of Dry Matter between Cytoplasm Fractions and CellSerum Plant Source Marigold Product Alfalfa Barley Lavender Flowers SageFresh Biomass 100.0 100.0 100.0 100.0 100.0 Cell Juice 32.7 28.5 28.137.6 21.9 Cell Juice Supernatant 20.5 15.6 19.4 28.1 15.2 CytoplasmFraction 2.3 0.4 0.8 0.9 0.2 Cell Serum 18.2 15.2 18.6 27.2 15.0

Table 22 shows that the yield of cytoplasm fractions dry matter did notexceed 2.5% of initial biomasses dry matter and subsequently 11% of celljuice supernatant dry matter. Most of cell juice supernatant dry matterwas concentrated in cell sera: 88.8% (alfalfa), 97.4% (barley), 95.9%(lavender), 96.8% (marigold flowers), and 98.7% (sage). Based on highdry matter yield, cell sera were selected as a prospective source forpreparation of soluble cosmetic ingredients.

Example 16 Optimum Composition of Preservatives and Antioxidants forCosmetic Botanical Ingredients From Alfalfa, Barley, Lavender, MarigoldFlowers, and Sage

The optimum composition of preservatives and antioxidant was determinedto be very similar for all plant sources (i.e., from alfalfa, barley,lavender, marigold flowers, and sage (see Table 23).

TABLE 23 Optimum Composition of Preservatives and Antioxidant (%)Required for Stabilization of Cell Serum Filtrates Plant Source MarigoldComponent Alfalfa Barley Lavender Flowers Sage Preservatives PotassiumSorbate 0.1 0.1 0.1 0.1 0.1 Sodium Benzoate 0.1 0.1 0.1 0.1 0.1 SodiumMethyl 0.1 0.1 0.1 0.1 0.1 Paraben Citric Acid — — 0.1 — 0.1Anti-oxidant Sodium Metabisulfite 0.2 0.2 0.2 0.1 0.2

Example 17 Comparison of Various Characteristics of Cosmetic BotanicalIngredients From Alfalfa, Barley, Lavender, Marigold Flowers, and Sage

The physico-chemical, spectral, microbial, toxicological, performanceand efficacy data related to cosmetic botanical ingredients arepresented in Tables 24 and 25.

TABLE 24 Physical and Chemical Properties of Cosmetic IngredientsProduced from Cell Serums Cosmetic Botanical Ingredient and Plant Source401 101 201 301 Marigold 501 Parameter Alfalfa Barley Lavender FlowersSage Solid Content, % 5.3 3.5 4.2 3.9 4.0 Specific Gravity, 1.025 1.0191.020 1.019 1.021 g/cm³ Color 6-7 5-6 11-12 4-5 8-9 Refractive Index1.342 1.338 1.341 1.340 1.340 pH 4.1 4.1 3.9 3.4 3.2 Red-Ox Potential,70 95 170 160 190 mV Conductivity, S/m 0.96 1.09 0.79 0.40 0.99

TABLE 25 UV-Spectral Properties of Cosmetic Ingredients Produced fromCell Serums Cosmetic Botanical Ingredient and Plant Source 401 101 201301 Marigold 501 Peak Parameter Alfalfa Barley Lavender Flowers Sage 1Start, nm 400.0 400.0 400.0 400.0 400.0 Apex, nm 324.5 335.5 260.5 260.0330.0 End, nm 303.0 305.0 236.5 232.5 306.5 Height, 0.347 0.232 2.4091.171 0.260 Abs Area, 21.197 14.254 135.505 55.719 14.952 Abs × nm 2Start, nm 303.0 305.0 — — 306.5 Apex, nm 258.0 258.0 — — 259.0 End, nm233.0 233.0 — — 235.0 Height, 1.471 1.268 — — 1.248 Abs Area, 65.10355.631 — — 57.844 Abs × nm

The data presented in Table 24 and Table 25 demonstrates that althoughfive plant source raw materials were used (even belonging to differentplant families), the properties of the cosmetic botanical ingredientsproduced from a variety of cell sera are very similar. This similaritycan be very valuable for manufacturing of highly standardized naturalproducts based on the cosmetic botanical ingredients described above.

The data presented in Table 26 demonstrate that all tested cosmeticbotanical ingredients satisfy the cosmetic industry requirement to theCFU level (colony forming units). The absence of pathogens alsosatisfied the industry requirements to the safety of cosmeticingredients.

TABLE 26 Microbial Data of Cosmetic Ingredients Produced from CellSerums Cosmetic Botanical Ingredient and Plant Source 401 101 201 301Marigold 501 Parameter Alfalfa Barley Lavender Flowers Sage Colony <100<100 <100 <100 <100 Forming Units (CFU) per gram of sample E. coli Nega-Nega- Nega- Nega- Nega- tive tive tive tive tive Candida Nega- Nega-Nega- Nega- Nega- albicans tive tive tive tive tive Pseudomonas Nega-Nega- Nega- Nega- Nega- sp. tive tive tive tive tive StaphylococcusNega- Nega- Nega- Nega- Nega- aureus tive tive tive tive tive

In addition, the cosmetic botanical ingredients demonstrated absence ofcytotoxicity in the wide concentration ranges. As an example, a testinvolving neutral red uptake (NRU) by 3T3 fibroblasts, which is commonlyused for determination of cytotoxicity, was utilized to assesscytotoxicity of cosmetic botanical ingredients. The NRU value isproportional to the number of viable cells in this in vitro testpopulation. It was found that 50% inhibition of neutral red uptake(NRU₅₀) by 3T3 fibroblasts was not reached even at very highconcentrations of tested cosmetic ingredients. As a comparison withrespect to the safety profile, most of the cosmetic botanicalingredients produced by the described methods were close to EpidermalGrowth Factor (EGF) as used as a positive control (see Table 27).

TABLE 27 Cytotoxicity Data of Cosmetic Ingredients Produced from CellSerums and Epidural Growth Factor (EGF) Cosmetic Botanical Ingredientand Plant Source 401 Positive 101 201 301 Marigold 501 Control ParameterAlfalfa Barley Lavender Flowers Sage EGFNRU₅₀, >2,500 >2,500 >400 >2,500 2,430 >2,500 μg Dry Matter/mL Cell NCMNCM MLT NCM NCM NCM Morphology* *NCM: Normal Cell Morphology; MLT:Moderate Level of Toxicity.

The cosmetic botanical ingredients produced from cell sera havesignificantly higher safety profile when compared with water extractisolated by conventional method from the same batch of dried rawmaterial or commercial extract obtained from the same plant source. Asan example, the data related to marigold flowers presented in the tablebelow (see Table 28).

TABLE 28 Cytotoxicity Data of Cosmetic Botanical Ingredient 402, WaterExtract and Commercial Extract Isolated from Marigold Flowers TestedProduct Cosmetic Botanical Ingredient Water Commercial Parameter 402Extract Extract EGF NRU₅₀, μg >2,500 41 2 >2,500 Dry Matter/mL CellMorphology* NCM HLT HLT NCM *NCM: Normal Cell Morphology; HLT: HighLevel of Toxicity.

All cosmetic botanical ingredients produced from cell sera demonstratedanti-oxidant properties or more specifically superoxide scavengingability. As an example, the concentrations required to inhibit 50% ofcytochrome c reduction (ICR₅₀) were determined and rosmarinic acid (RA)was used as a positive control (see Table 29).

TABLE 29 Superoxide Scavenging Ability of Cosmetic Ingredients Producedfrom Cell Serums and Rosmarinic Acid Cosmetic Botanical Ingredient andPlant Source Positive 401 Control 101 201 301 Marigold 501 RosmarinicAlfalfa Barley Lavender Flowers Sage Acid ICR50, 149 160 158 153 16026.5 μg Dry Matter/mL

Example 18 Characterization of Cosmetic Ingredients Produced fromMembrane Fraction

The physico-chemical, optical, microbial, toxicological, performance andefficacy data related to selected cosmetic ingredients produced frommembrane fractions are presented in Tables 30 and 31.

TABLE 30 Physico-Chemical Properties of Cosmetic Ingredients Producedfrom Membrane Fractions Cosmetic Botanical Ingredient and Plant Source401 502 Parameter Marigold Flowers Sage Non-Volatile Residue, % 7.1 8.3Specific Gravity, g/cm³ 1.054 1.047 Viscosity, cps 15,800 5,200 pH 4.64.6

TABLE 31 L*a*b* Values of Cosmetic Ingredients Produced from MembraneFractions Cosmetic Botanical Ingredient and Plant Source 402 502Marigold Flowers Sage L* 33.27 27.35 a* 20.36 −1.4 b* 49.56 16.97

The data presented in Table 30 and Table 31 demonstrate significantdifferences between properties of cosmetic ingredients produced frommembrane fractions obtained from leaf-and-stalk biomass (sage) and fromflowers (marigold). Generally, the above reflects the difference betweenchloroplasts, which are predominantly concentrated in sage membranefraction, and chromoplasts, which are predominantly concentrated inmarigold flowers.

Microbial analyses demonstrated that Cosmetic Botanical Ingredients 402and 502 satisfy the cosmetic industry requirements for cosmeticingredients with regard to CFUs and absence of pathogens (see Table 3,above, for methods).

Example 19 Anti-Inflammatory and Antioxidant Analysis of CosmeticBotanical Ingredients: Objectives and Rationale for SelectedExperimental Models

Cosmetic Botanical Ingredients 101, 102, 201, 202, 301, 302, 401, 402,501, and 502, as well as others, were analyzed for theiranti-inflammatory and antioxidant qualities. The results of theseanalyses are summarized in this Example 19, below. The procedures andresults are explained in Examples 20-24, below.

The described procedure pertains to the distribution of concentratedserum-derived and membrane-derived cosmetic botanical ingredients. Theseingredients demonstrated two important activities (antiproteolyticactivity and antioxidant activity) towards reducing connective tissuedamage associated with inflammation. The pattern of distribution forantiproteolytic activity is selectively in the membrane fractions andsubsequently in multiphase botanical cosmetic ingredients, where as thedistribution pattern for antioxidant activity is selectively in theserum fraction and subsequently soluble in cosmetic ingredients.Membrane-derived cosmetic ingredients contain components which inhibitboth of the two major classes of destructive proteinases, i.e., serineproteinases exemplified by neutrophil elastase and matrixmetalloproteinases exemplified by gelatinase B. The potential of themembrane-derived cosmetic ingredients to achieve inhibition of thesynergistic proteolytic activities of inflammatory cells meritsconsideration of their use in topical applications for anti-inflammatoryformulations. The mode of inhibition of these cosmetic ingredientssuggests that their effects are reversible, and, they would not causeundesirable long term modifications to defense or repair mechanisms.

The selective distribution of antioxidant activities into theserum-derived cosmetic ingredients presents an additional direction toincorporate an important biological activity which reduces damage causedby the reactive oxygen species generated by inflammatory cells. Theserum-derived cosmetic ingredients obtained from multiple botanicalsources possess potent modulatory activities which diminish the capacityof the inflammatory cells to generate reactive oxygen species ratherthan simply neutralizing the oxidants. The described method employed ingeneration of the serum-derived cosmetic ingredients result in thepreservation of this modulatory activity along with scavenging activity.The conventional procedures for obtaining aqueous extracts simplyachieve only some distribution of scavenging activity alone.

The selective distribution of one type of biological activity into themembrane-derived cosmetic ingredient and another activity into theserum-derived cosmetic ingredient obtained from the same botanicalsources represents an opportunity to employ novel topical formulationsin which two phases are maintained in stable composition.

Cosmetic Botanical Ingredients 101, 201, 301, 401, 402, 501, and 502(collectively referred to herein as the “Cosmetic BotanicalIngredients”) were evaluated for their anti-inflammatory and antioxidantactivities. There are multiple mechanisms for injury to connectivetissue that may arise as a consequence of the inflammatory process. Theone final common pathway leading to inflammatory tissue injury involvesdestruction of the components of the stroma by white blood cell-derivedproteolytic enzymes. Accordingly, assays were employed to evaluate thecapacity of the different Cosmetic Botanical Ingredients to inhibitthese inflammatory proteinases. In the evaluation, two proteinases wereused: neutrophil elastase and neutrophil gelatinase. These two enzymesdegrade the components of the extracellular matrix of human connectivetissue in a synergistic manner. Moreover, neutrophil elastase caninactivate the body's own inhibitory defenses against neutrophilgelatinase while conversely, the gelatinase can inactivate the body'sown antielastase defenses. Thus, cosmetic botanical ingredients whichcan inhibit these two enzymes provide significant protection againstinflammatory injury. The assays selected permit quantitation of theinhibitory activity of these cosmetic botanical ingredients and provideinformation regarding some basic features of the mode of inhibition.

In addition to degradative proteinases, inflammatory processes are oftenassociated with release of reactive oxygen species from the activatedcells. These reactive species include superoxide anions, hydroxylradicals, hydrogen peroxide, and hypochlorous acid. The biologicaleffects of these oxidants can lead to inactivation of importantendogenous antiproteolytic defenses in the human tissue. Assays wereemployed which measure the capacity of the cosmetic botanicalingredients to lower the levels of reactive oxygen species released byactivated inflammatory cells. Additionally, assays were used toquantitate the capacity of the cosmetic botanical ingredients toneutralize reactive oxygen species of endogenous and exogenous origins.

Example 20 Evaluation of Anti-Elastase Activity

During the inflammatory process, elastase activity is directly relatedto the actions of multiple enzymes, but neutrophil elastase is presentedat the highest concentrations and is the most active proteinase againstthe widest variety of connective tissue components, including elastin.In the evaluation assay, inhibition of this enzyme employed a syntheticsoluble peptide substrate(Methoxysuccinyl-Ala-Ala-Pro-Val-p-Nitroanilide) that is specific forneutrophil elastase. The source of neutrophil elastase was a purifiedenzyme preparation derived from the airway secretions of patients withcystic fibrosis. Analysis of the concentration dependence of inhibitionleads to the quantitation of potency of the inhibitory activity. Thisactivity is expressed as that concentration of dry matter within eachcosmetic botanical ingredient required to achieve 50% inhibition (IC₅₀).In addition, the value of the inhibition constant, K_(i), wasdetermined. Graphical analysis of the inhibition data also provides theinformation related to the mode of inhibition (reversible orirreversible). Since neutrophil elastase has positive physiologicalroles when present at controlled levels, indiscriminate use ofirreversible inhibitors may compromise these normal functions of theenzyme.

Table 32 describes the results of the in vitro elastase inhibitionstudies of the serum-derived and membrane-derived cosmetic ingredients.

TABLE 32 Elastase Inhibition Evaluation of Cosmetic BotanicalIngredients IC₅₀ K_(i) Cosmetic Botanical Ingredient (Source) μg/mlμg/ml 101 (Alfalfa Serum Fraction) No No Inhibition Inhibition 102(Alfalfa Membrane Fraction) 9.5 7.2 201 (Barley Serum Fraction) No NoInhibition Inhibition 202 (Barley Membrane Fraction) 4.6 3.6 301(Lavender Serum Fraction) 36.0 25.4 302 (Lavender Membrane Fraction)10.0 2.3 401 (Marigold Flowers Serum Fraction) No No InhibitionInhibition 402 (Marigold Flowers Membrane Fraction) 21.0 0.68 501 (SageSerum Fraction) 115.0 70.3 502 (Sage Membrane Fraction) 30.0 12.0Positive Control (Elhibin ®) 4.0 3.4

Elastase inhibition activity has been identified predominantly incosmetic ingredients obtained from membrane fractions. Cosmeticingredients produced from alfalfa, barley and marigold flowers serumfractions did not display any elastase inhibition. Cosmetic ingredientsobtained from lavender and sage serum fractions did show much lowerinhibitory activity compared with corresponding cosmetic ingredientsobtained from membrane fractions. The above pattern of distribution ofelastase inhibition activity between the membrane-derived andserum-derived cosmetic botanical ingredients was found for all testedraw material sources.

Selected cosmetic ingredients obtained from membrane fractionsdemonstrate elastase inhibition activity which is comparable inmagnitude with the activity of a specific elastase inhibitor used as apositive control.

Cosmetic ingredients obtained from membrane fractions demonstratedproperties consistent with “classical” simple competitive and reversibleelastase inhibitors, while the positive control has a complex inhibitorybehavior (including some irreversible inhibitory activity).

The inhibitory properties of cosmetic ingredients produced from membranefractions toward the most destructive inflammatory proteinase(neutrophil elastase) qualifies these ingredients as valuable componentsof topical products for use as anti-inflammatory agents.

Example 21 Elastase Inhibition Evaluation of the Marigold Products

The in vitro elastase inhibition evaluation of marigold products aredescribed below and summarized in Table 33.

TABLE 33 Elastase Inhibition Evaluation of Marigold Products CosmeticBotanical Ingredient (Source) IC₅₀ K_(i) and Extract μg/ml μg/ml 401(Marigold Flowers Serum Fractions) No No Inhibition Inhibition 402(Marigold Flowers Membrane Fractions) 21.0 0.68 Commercial Extract(Marigold Flowers) 120.0 152.0 Commercial Extract (Marigold Flowers) NoNo Inhibition Inhibition

indicates data missing or illegible when filed

Cosmetic ingredient 402, obtained from marigold flowers membranefraction, demonstrated the highest elastase inhibition activity, but thecosmetic ingredient 401 obtained from serum fraction, which was derivedfrom the same raw material and separated from membrane fraction duringcell juice fractionation process, has no detectable inhibition activity.

The water extract which was produced by conventional extraction methodswas obtained from the same batch of raw material and did not displayelastase inhibition activity.

The commercial extract, which was derived from same raw material,displayed only minimal anti-elastase activity.

The cosmetic ingredient 402 derived from same raw material displayedanti-elastase activity by about two orders of magnitude over that of thecommercial extract.

Example 22 Evaluation of Anti-Gelatinase Activity of the CosmeticBotanical Ingredients

Neutrophils contain two major enzymes from the class of matrixmetalloproteinases, which collectively are implicated in extensiveconnective tissue destruction: neutrophil collagenase (MMP-8) andgelatinase B (MMP-9). Because elastase has poor activity against nativecollagen, and neutrophil collagenase alone cannot solubilize theconnective tissue protein by itself, gelatinase B is considered as amajor contributor to inflammatory injury to the extracellular matrix. Aspecific assay for this enzyme was used to evaluate the potential ofcosmetic botanical ingredients to inhibit degradation of theextracellular matrix mediated by inflammatory cell-derived matrixmetalloproteinases. Gelatinase B activity was detected by hydrolysis ofa low molecular weight synthetic substrate (APMA) Inhibitors ofgelatinase B diminish the accelerating rate of enzyme reaction productformation in a dose-dependent fashion. Such enzyme inhibition was foundwhen tested cosmetic ingredients were added to the reaction mixture.

The anti-gelatinase B data regarding the Cosmetic Botanical Ingredientsis described below and summarized in Table 34.

TABLE 34 In Vitro Gelatinase B Inhibition Evaluation of CosmeticBotanical Ingredients. % Inhibition Cosmetic Botanical Ingredient(Source) [100 μg/ml] 101 (Alfalfa Serum Fraction) 20 201 (Barley SerumFraction) 32 301 (Lavender Serum Fraction) 21 401 (Marigold FlowersSerum Fraction) 42 501 (Sage Serum Fraction) 29 502 (Sage MembraneFraction) 100 Positive Control (Rosmarinic Acid) 91

All serum-derived cosmetic botanical ingredients have demonstratedmodest gelatinase B inhibition activity.

Cosmetic Botanical Ingredient 502 obtained from the sage membranefraction demonstrated significant gelatinase B inhibition activity,which exceeded by at least three times the corresponding activity of theserum-derived cosmetic ingredient. The pattern of distribution ofgelatinase B inhibitory activity between membrane-derived andserum-derived cosmetic ingredients was similar to the distributionpattern found for anti-elastase activity.

Cosmetic Botanical Ingredient obtained from sage membrane fractiondemonstrated potent gelatinase B inhibition activity (IC₅₀=24.9 μg/ml)comparable to that of a positive control (rosmarinic acid) havingIC₅₀=30 μg/ml.

The inhibitory properties of membrane-derived Cosmetic BotanicalIngredients toward gelatinase B indicate that this ingredient has valueas an active component of anti-inflammatory topical products.

Example 23 In Vitro Evaluation of Suproxide Scavenging Activity for theCosmetic Botanical Ingredients

Reactive oxygen species generated by activated inflammatory cells(endogenous or exogenous) create specific oxidant which was used toprovide the measurements of superoxide scavenging activity. The assayused for evaluation of superoxide scavenging activity relates to the oneform of antioxidant activity, which is of benefit in neutralizing thedamage associated with oxidation. To generate superoxide anions in highyield and in a controlled fashion, an enzymatic system (xanthineoxidase) was used. The conversion of xanthine to hypoxanthine by thisenzyme generates amounts of superoxide anions, which are stoichiometricwith the amount of substrate provided. The assay used was based on thereduction of cytochrome c from its ferric to ferrous form as a sensitivemeasure of superoxide levels. The advantage of using cytochrome creduction to detect superoxide anions generated by the action ofxanthine oxidase on xanthine is that the same measure may be employed todetect the release of superoxide anions by activated inflammatory cellsundergoing a “respiratory burst.” Cosmetic Botanical Ingredients whichdecrease the magnitude of the respiratory burst but do not scavengeenzymatically generated superoxide anions are presumably inhibiting someaspect of cell function rather than acting as scavengers of the reactiveoxygen species generated by the cells.

The superoxide scavenging data is described below and Tables 35 and 36.

TABLE 35 Evaluation of Superoxide Scavenging Activity of CosmeticBotanical Ingredients. IC₅₀ Cosmetic Botanical Ingredient (Source) μg/ml101 (Alfalfa Serum Fraction) 149 102 (Alfalfa Membrane Fraction) NoInhibition 201 (Barley Serum Fraction) 160 202 (Barley MembraneFraction) No Inhibition 301 (Lavender Serum Fraction) 158 302 (LavenderMembrane Fraction) No Inhibition 401 (Marigold Flowers Serum Fraction)153 402 (Marigold Flowers Membrane Fraction) No Inhibition 501 (SageSerum Fraction) >160 502 (Sage Membrane Fraction) No Inhibition PositiveControl (Rosmarinic Acid) 26.5

Superoxide scavenging ability is fully concentrated in serum-derivedcosmetic botanical ingredients. Membrane-derived cosmetic botanicalingredients did not demonstrate any superoxide scavenging ability. Theabove distribution pattern of superoxide scavenging ability was foundfor all tested raw material sources.

Serum-derived cosmetic botanical ingredients demonstrated approximately20% of the superoxide scavenging ability of the positive control(rosmarinic acid).

The superoxide scavenging ability of the serum fractions suggest thatthese cosmetic botanical ingredients have value as prospectivecomponents to act as topical antioxidant and UV-protectant products.

TABLE 36 Evaluation of Superoxide Scavenging Activity of MarigoldProducts IC₅₀ Cosmetic Botanical Ingredient or Extract μg/ml 401(Marigold Flowers Serum Fractions) 153 402 (Marigold Flowers MembraneFractions) No Inhibition Commercial Extract (Marigold Flowers) >160Conventional Extract (Marigold Flowers) 53

Marigold flowers serum-derived Cosmetic Botanical Ingredientdemonstrated significant superoxide scavenging ability, but themembrane-derived Cosmetic Botanical Ingredient (derived from the sameraw material) has no detectable inhibition ability.

Commercial extract, which was derived from the same raw material,displayed superoxide scavenging ability which was comparable to that ofthe serum-derived Cosmetic Botanical Ingredient.

The water extract isolated by conventional methods from the same batchof raw material demonstrated higher superoxide scavenging ability thanthe serum-derived Cosmetic Botanical Ingredient or the commercialextract.

Example 24 Evaluation of the Effect of Cosmetic Botanical Ingredients onNeutrophil Respiratory Burst

The superoxide scavenging activity of different cosmetic botanicalingredients described in the previous Example 23 was measured with an invitro enzymatically generated source of superoxide anions, and with anin vivo reactive oxygen species generated by activated inflammatorycells (i.e., neutrophils). Neutrophils are especially important sourcesof reactive oxygen species, because they are involved in greatestnumbers to sites of local inflammation and because they convert some ofthe species, such as superoxide anions and hydrogen peroxide to anantioxidant such as hypochlorous acid. Detection of the superoxideanions which are released into the extracellular environment byneutrophils is a sensitive measure of the overall levels of activity ofthese cells to generate multiple reactive oxygen species, collectivelyreferred to as the “respiratory burst.” The same reagent was employed todetect the extracellular superoxide derived from neutrophils as used todetect superoxide formed enzymatically, i.e., ferricytochrome c. Becausethis molecule is a protein and cannot enter the neutrophil, it does notdetect intracellular reactive oxygen species.

Phorbol myristate acetate (PMA), which is known to mimic the signals forat least two independent pathways for neutrophil activation, was used asa stimulant of respiratory burst in vivo. The rate of cytochrome creduction by the PMA-activated neutrophils is proportional to themagnitude of the respiratory burst in these cells. Results ofdose-dependent inhibition were expressed in terms of the maximal rate ofcytochrome c reduction observed after a 150 second lag phase followingaddition of PMA.

A review of the data regarding the inhibition of neutrophil respiratoryburst studies are presented in Table 37.

TABLE 37 Neutrophil Respiratory Burst Evaluation of Cosmetic BotanicalIngredients. Cosmetic Botanical V_(max) Ingredient (Source) 0 0.5 25.050.0 Dose Response and Extract μg/ml μg/ml μg/ml μg/ml Effect Control(Unstimulated)  1.96 — — — — Control (Stimulated) 18.86 — — — — 501(Sage Serum — 12.11 11.0 19.0 Inhibition/ Fraction) Stimulation 401(Marigold Flowers —  9.11 8.79 19.48 Inhibition/ Serum Fraction)Stimulation Commercial Extract — 15.15 11.29 10.33 Only Inhibition(Marigold Flowers) Conventional Extract — 21.26 10.33 8.82 OnlyInhibition (Marigold Flowers)

Both tested Cosmetic Botanical Ingredient 401 and 501 demonstratedbiphasic modulation of the respiratory burst from PMA-stimulatedneutrophils. At low concentrations, the serum-derived cosmetic botanicalingredients exhibited strong inhibitory activity, but at highconcentrations this inhibitory activity was replaced by net modeststimulation of the respiratory burst above that of neutrophilsstimulated with PMA alone. Therefore, serum-derived cosmetic botanicalingredients contain components having a stimulatory effect onneutrophils, but these components have only moderate potency, asstimulation is observed only at high concentrations. In addition, theserum-derived cosmetic botanical ingredients contain other componentswhich inhibit the respiratory burst at very low concentrations. Theinhibition of the neutrophil respiratory burst at these lowconcentrations (˜2.5 μg dry material/ml) cannot be due simply tosuperoxide scavenging activity, which required much higherconcentrations of dry material (˜150 μg/ml) to be detected.

Commercial extract, which was derived from marigold flowers, did notdisplay any stimulatory effect and demonstrated only inhibition ofrespiratory burst activity.

Water extract isolated by conventional methods from the same batch ofmarigold flowers as the serum-derived cosmetic botanical ingredient, didnot display stimulatory activity. The conventional extract did notretain any of the extremely potent inhibitory activity towards theneutrophil respiratory burst seen at very low (2.5 μg/ml) concentrationsof the serum-derived cosmetic botanical ingredients.

Example 25 Protocols Used for Determining Certain Characteristics ofCosmetic Botanical Ingredients 101, 201, 301, 401, 402, 501, and 502

The following are various methods used for determining certaincharacteristics of Cosmetic Botanical Ingredients 101, 201, 301, 401,402, 501, and 502. These methods are referenced throughout the aboveExamples. References below to the “tested products” or the “testsamples” refer to Cosmetic Botanical Ingredients 101, 201, 301, 401,402, 501, and 502.

Method 1: Method for Determination of Solid Content.

The procedure for determination of solid content included evaporation ofthe tested product in the water bath at 100° C. until completeevaporation of water, oven storage of the sample at 105° C. for 3 hours,cooling to room temperature, and immediate determination of the weightof the container with solid matter.

Method 2: Method for Determination of Non-Volatile Residue.

The procedure for determination of non-volatile residue included ovenstorage of the tested product at 105° C. for 5 hours, cooling, andimmediate determination of the weight of the container with solidmatter.

Method 3: Method for Determination of L*a*b* Values.

The procedure for determination of L*a*b* values utilized Hunter Labscanfixed geometry calorimeter with measuring geometry of 0°/45°. Standardilluminant D₆₅ with viewing window facing upward was used. The containerwith tested product was placed on viewing window and measured throughthe bottom. The following CIELAB equations were used:

C*=(a* ² +b* ²)^(1/2)

DE*=[(DL)²+(Da*)²+(Db*)²]^(1/2)

DH=[(DE*)²−(DL*)²−(DC*)²]^(1/2)

Method 4: Method for Determination of Total Carotenoids Content andLutein Content.

The tested samples were extracted with acetone. After homogenization andvacuum filtration, all extracts were saponified with 30% potassiumhydroxide in methanol. The carotenoids were successively extracted withpetroleum ether. After additional treatment and re-solubilization inethanol, all samples were measured at 446 nm.

In order to determine the lutein content, an additional dried samplefrom each sample extraction was used for HPLC analysis. The dried samplewas re-solubilized in MTBE and methanol. The reverse phase HPLC systemwith (250×4.60 mm I.D.) 5 μm C₁₈ column (“Vydac”) was used. The identityof lutein was conformed by the co-chromatography of an authenticstandard. The molar absorptivity coefficient for lutein in ethanol is144,800 cm⁻¹ mol⁻¹.

Method 5: Method for Determination of Elastase Inhibitory Activity.

The elastase inhibitory activity of tested fractions was determinedusing the assay, which employs neutrophil elastase (a purified enzymepreparation produced by “Elastin Products”) and synthetic peptidesoluble substrate Methoxysuccinyl-Ala-Ala-Pro-Val-p-Nitroanilideproduced by “Sigma”. Enzymatic cleavage of the substrate results ingeneration of increasing yellow color over time (405 nm); the rate ofcolor generation is diminished by increasing concentrations of testedfractions containing inhibitory activity. Analysis of the concentrationdependence of inhibition permits quantitation of the potency of theinhibitory activity, expressed as that concentration of dry matterwithin each tested fraction required to achieve 50% inhibition (IC₅₀),but also provides information relating to the mode of inhibition.

For the determination of IC₅₀, the concentration of elastase was 2.5μg/ml and concentration of substrate was 150 μM. For the determinationof K_(i), the concentrations of substrate were 100 μM and 200 μM.

Method 6: Method for Determination of Gelatinase B Inhibitory Activity.

The commercially distributed assay (MMP-9 Activity ELISA produced by“Amersham Pharmacia”), which captures Gelatinase B specifically ontomultiwell microplates by immune recognition, was used after otherproteinases were washed away. The enzymatic activity was detected at 405nm by hydrolysis of a low molecular weight synthetic substrate forGelatinase B: APMA. Analysis of the concentration dependence ofinhibition was used to determine the potency of tested product drymatter.

Method 7: Method for Determination of Superoxide Scavenging Activity.

The enzymatic system, which uses xanthine oxidase (a purified enzymepreparation produced by “Sigma”), was used to generate superoxide anionsin high yield and in a controlled fashion. The conversion of xanthine tohydroxanthine by this enzyme generates amounts of superoxide anions andreduction of ferricytochrome c to ferrocytochrome c was used as asensitive measure of superoxide levels. The measurements offerrocytochrome c level (550 nm), when tested fractions were added tothe reaction system, allow for determination of their superoxidescavenging activity. The final concentrations per well were forcytochrome c 75 μM, xanthine 425 μm/L, and xanthine oxidase 10 mU/ml.

Method 8: Method for Determination of Inhibition of the NeutrophilRespiratory Burst.

The phorbol myristate acetate (PMA produced by Alexis Corporation, SanDiego, Calif.) was used as a trigger of the respiratory burst activitydemonstrated by neutrophils. The detection of superoxide anions, whichare released into the extracellular environment by neutrophils, wasachieved via measurements of ferrocytochrome c level. The rate ofcytochrome c reduction by PMA-activated neutrophils is proportional tothe magnitude of the respiratory burst in these cells. Results ofdose-dependent inhibition were expressed in terms of maximal rate ofcytochrome c reduction observed at 550 nm after 150 seconds lag phasefollowing addition of PMA.

Example 26 Protocols Used for Determining Certain Characteristics ofVarious Cosmetic Botanical Ingredients of the Present Invention,Including Cosmetic Botanical Ingredients 601 and 701

The following are various methods used for determining certaincharacteristics of various cosmetic botanical ingredients of the presentinvention, including, without limitation, Cosmetic Botanical Ingredients601 and 701. These methods are referenced throughout the Examples. Inparticular instances, references below to the “tested products” or the“test samples” refer to Cosmetic Botanical Ingredients 601 and/or 701.

Osmolality

Osmolality is the measure of solute concentration, defined as the numberof osmoles of solute per kg of solution. Osmolality measures the numberof osmoles of solute particles per unit mass of solution. Osmolality isdistinct from molarity because it measures moles of solute particlesrather than moles of solute. The distinction arises because somecompounds can dissociate in solution, whereas others cannot. Ioniccompounds, such as salts, can dissociate in solution into theirconstituent ions, so there is not a one-to-one relationship between themolality and the osmolality of a solution. For example, sodium chloride(NaCl) dissociates into Na+ and Cl− ions. Thus, for every 1 mole of NaClin solution, there are 2 osmoles of solute particles (i.e., a 1 M NaClsolution is a 2 Osm NaCl solution). Both sodium and chloride ions affectthe osmotic pressure of the solution. Nonionic compounds do notdissociate, and form only 1 osmole of solute per 1 mole of solute. Forexample, a 1 M solution of glucose is 1 Osm (Widmaier, Eric P.; HershelRaff, Kevin T. Strang (2008). Vander's Human Physiology, llth Ed.McGraw-Hill. pp. 108-112). Osmometer, model 3250 (Advanced Instruments,Inc) was used to determine osmolalities of the ingredients. Thisinstrument utilizes freezing point depression as measuring principle.Freezing point is a colligative property that is dependent on thepresence of dissolved particles and their number, but not theiridentity. The freezing point depression happens both when the solute isan electrolyte, such as various salts, and a non-electrolyte, such ascarbohydrates.

Dry Matter

Dry matter reflects the concentration of non volatile components in theingredients. Dry matter levels were determined by comparing the weightof liquid sample with weight of residual dry matter after liquidcomponents have been evaporated. Disposable aluminum weighing dishes(VWR 25433-016), Ohaus Explorer E00640 balance (Ohaus Corporation) andShel Lab model 1400E oven (VWR) set at 105 C were utilized. Dry matterpercentage is calculated as (‘tare+dry’−‘tare’)/(‘tare+wet’−‘tare’)*100.

Color (Gardner Scale)

The Gardner Color scale as specified in ASTM D1544 is a single numbercolour scale for grading light transmitting samples with colorcharacteristics ranging from light yellow to brownish red. The scale isdefined by the chromaticities of glass standards numbered from 1 for thelightest to 18 for the darkest. The Gardner Color of samples wasdetermined on the Lovibond Gardner Comparator 3000 (The TintometerLimited of Salisbury, UK), a 3-field instrument for visually determiningthe Gardner Color of samples by direct comparison with colored glassstandards.

Refractive Index, nD

Refractive Index was determined by measuring on Arias 500 refractometerfrom Reichert Analytical Instruments (Depew, N.Y.) with temperatureregulation provided by Polystat model 12108-10 temperature controllerfrom Cole-Parmer (Vernon Hills, Ill.). Procedure is based on theinstruction manual for Arias 500 refractometer, sections 6.0, 4.1 and4.4-4.5.

pH Determination

pH is defined as minus the decimal logarithm of the hydrogen ionactivity in a solution and used to determine pH is a measure of theacidity or basicity of a solution.

pH levels were determined on a pH meter Model 250 pH/ISE/conductivitymeter from Denver Instrument Company (Bohemia, N.Y.) with pH/ATCelectrode number 300729.1 (Denver Instrument Company). Procedure isbased on Denver Instrument Company 301127.1 Rev. D manual, pages ii and9-12.

Lambda Max, nm

Lambda max, nm was determined on Ultrospec 4300 pro UV/Visiblespectrophotometer from Biochrom Ltd (Cambridge, UK), formerly under GEHealthcare, formerly known as Amersham Biosciences, with water heatedcell holder (Amersham part #80-2106-08). The procedure is based onsections 2 and 4 from Amersham manual number 80-2108-25 entitled SWIFTII Applications Software UV/Visible Spectrophotometers, and on pages 7and 15 from Amersham manual number 80-2111-79 entitled Ultrospec 4300pro UV/Visible Spectrophotometer User Manual. Instrument control wasprovided by SWIFT II software suite (Biochrom Ltd) and temperatureregulation by CB20 Mini Circulator from Torrey Pines Scientific(Carlsbad, Calif.).

Determination of Protein

The Kjeldahl method was used to measure the protein nitrogen content.

Microbiological Limits

Microbial content and limits: Total Plate Count, CFU/g; Mold and Yeast,CFU/g; E. coli; Salmonella sp.; Staphylococcus aureus; Pseudomonas sp.were determined according to US Pharmacopoeia XXX, NF25, <61>,Microbiological Limit Tests.

Trypsin Inhibition

Trypsin is a proteolytic enzyme that is involved in in vivo epidermalproliferation and inflammation. Trypsin inhibition activity wasdetermined by a kinetic colorimetric assay designed for use with 96-wellmicrotiter plates (microplates) and computer-controlled microplatereader. Enzymatic activity in cleaving the substrate was indicated by adevelopment of yellow color measured as increase in absorbance at 405 nmwavelength. The mean of maximum rates of absorbance increase fornegative control wells was considered as 100% of enzyme activity, andIC₅₀ was calculated as concentration of sample in the well necessary toreduce the enzyme activity to 50%. Lower IC₅₀ values indicate highertrypsin inhibition activity. L-BAPA (Nα-Benzoyl-L-arginine4-nitroanilide hydrochloride) substrate, trypsin, and solvent reagentswere obtained from Sigma-Aldrich. pH 8.2 Tris-CaCl₂ buffer was used forpreparing working solutions of trypsin and L-BAPA substrate. Deionizedwater was used as solvent for buffer reagents, as negative control, andas the diluents for preparing serial dilutions of the samples. Reactionvolume in each well was 200 μl, with concentration of trypsin equal to60 nM and substrate equal to 0.5 mM.

Tyrosinase Inhibition

Tyrosinase is a copper-containing monooxygenase catalyzing theo-hydroxylation of monophenols to the corresponding catechols(monophenolase or cresolase activity), and the oxidation of monophenolsto the corresponding o-quinones (diphenolase or catecholase activity).These functions of tyrosinase play an important role in the formation ofmelanin pigments during melanogenesis. Melanin production is principallyresponsible for skin color and plays an important role in prevention ofsun-induced skin injury. However, abnormal accumulation of melaninproducts in skin is responsible for hyperpigmentations includingmelasma, chloasma, freckles, and senile lentigines, which can lead to anundesired aesthetic appearance (Jeon et al. (2005) Bull. Korean Chem.Soc, Vol. 26: 1135-1 137).

Tyrosinase is an important enzyme in the biosynthesis of melanin.Tyrosinase inhibition assay was used to search for ingredients that caninterfere with the ability of mushroom tyrosinase enzyme to convertL-tyrosine to L-dihydroxyphenylalanine (L-DOPA).

Tyrosinase inhibition activity was determined by a kinetic colorimetricassay designed for use with 96-well microtiter plates (microplates) andcomputer-controlled microplate reader. Enzymatic activity in convertingthe L-tyrosine substrate to L-DOPA (L-3,4-dihydroxyphenylalanine) wasindicated by a development of brown color measured as increase inabsorbance at 475 nm wavelength. The mean of maximum rate of absorbanceincrease for negative control wells was considered as 100% of enzymeactivity, and IC₅₀ was calculated as concentration of sample in the wellnecessary to reduce the enzyme activity to 50%. Lower IC₅₀ valuesindicate higher tyrosinase inhibition activity. L-tyrosine substrate andmushroom tyrosinase were obtained from Sigma. 1× pH 7.4 PBS (PhosphateBuffered Saline) buffer solution was obtained from Gibco. PBS was usedfor preparing working solutions of mushroom tyrosinase and L-tyrosinesubstrate. Deionized water was used as negative control and as diluentfor preparing serial dilutions of the samples. Reaction volume in eachwell was 200 mL, with concentration of mushroom tyrosinase equal to 13units/mL and L-tyrosine substrate equal to 0.5 mM.

LDH (Lactate Dehydrogenase) Release Cytotoxicity Method

LDH (lactate dehydrogenase) release cytotoxicity determination method isbased on the fact that certain cell component substances are typicallysequestered inside the cells and scarce in extracellular medium. Loss ofcell viability leads to loss of cell membrane integrity and release ofsuch substances. Extracellular LDH concentration can be measuredcolorimetrically with use of a dye which is converted by LDH intocolored form. Cytotoxicity of a test article can therefore be determinedby adding the test article to growth medium of a cell culture, measuringLDH concentration in the medium after proper incubation time, andcomparing the results with those obtained from growth medium of negativecontrol cell culture of healthy untreated cells, as well as those frompositive control cell culture treated with a known cytotoxic agent.

MatTek MelanoDerm Assay

MatTek Melanoderm Assay determines effect of a test article onmelanin-related skin pigmentation (such as for whitening/lighteningapplications). The assay is based on macroscopic and microscopicobservations and endpoint melanin content determination in a co-cultureof normal human keratinocytes and melanocytes that models humanepidermis and its color development. The Melanoderm cell cultures aremaintained and observed for the duration of the study, with untreatednegative controls compared to positive controls which have knownmelanogenesis-affecting substances added to growth medium, as well ascultures which have the test article added to growth medium. At the endof the study, the cell cultures are harvested and processed to measuremelanin content using a colorimetric assay.

Antioxidant Activity

Antioxidant is an agent that reduces the damage caused by oxidation.Antioxidant activity was determined by ORAC testing using an adaptationof the method described in “Performing Oxygen Radical AbsorbanceCapacity (ORAC) Assays with Synergy HT Multi-Detection MicroplateReader” Application Note from BioTek available at(www.biotek.com/resources/docs/ORAC_Assay_Application_Note.pdf) for usewith Synergy 2 microplate reader from BioTek Instruments Inc (Winooski,Vt.). In this assay, AAPH (2,2′-azobis 2-amino-propane) generatesreactive oxygen species which damage the fluorescent probe (sodiumfluorescein). Antioxidants such as (R)-Trolox methyl ether prevent orslow this damage, and their effects can be quantified by fluorescencemeasurements. Fluorescence readings were taken with excitationwavelength set at 485 nm and emission wavelength set at 528 nm, withreaction volume of 200 μA, AAPH concentration of 55 mM, sodiumfluorescein concentration of 1.33 μM, and (R)-Trolox methyl etherconcentration range between 80 μM and 2 μM. Sodium fluorescein (Fluka46960), AAPH (Sigma 440914) and (R)-Trolox methyl ether (Fluka 93509)were obtained from Sigma-Aldrich (St. Louis, Mo.). AUC (Area UnderCurve) values were calculated as sum of proportions (currentfluorescence reading for the well divided by first fluorescence readingfor the well). Average of AUC values of wells with deionized water wassubtracted from AUC of wells with (R)-Trolox methyl ether and wells withtest articles to obtain AUC corresponding to preservation offluorescence by antioxidants. A calibration curve was generated asfunction of a wells' antioxidant-related AUC showing (R)-Trolox methylether weight-equivalent ORAC activity. ORAC activity for test articleswas then calculated as units weight test article necessary to achieveantioxidant effect equal to one produced by 1 unit weight (R)-Troloxmethyl ether, with lower numbers indicating higher ORAC activity.

DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Free Radical Scavenging Activity

Free radical scavenger is an ingredient that reacts with free radicalsin a biological system, reduces free radical-induced damage, andprotects against the effects of free radicals. Free radical scavengingactivity, i.e. DPPH (2,2-Diphenyl-1-Picrylhydrazyl) free radicalscavenging activity, was determined by a kinetic colorimetric assayadapted for use with glass-coated polypropylene 96-well microtiterplates (catalog number 400 062) from SUN-SR1 (Rockwood, Tenn.) andSynergy 2 microplate reader from BioTek Instruments Inc (Winooski, Vt.).Absorbance was measured at 515 nm wavelength. Reaction volume in eachmicroplate well was 200 μA, with initial concentration of DPPH equal to114 μM. L-ascorbic acid was used as positive control. DPPH (Sigma D9132)and USP L-ascorbic acid (Sigma A-2218) were obtained from Sigma-Aldrich(St. Louis, Mo.). Stoichiometry of the reaction was calculated andexpressed as units weight test article necessary to quench 1 unit weightDPPH, with lower numbers indicating higher activity. This method wasadapted from procedure described in (“Use of a free radical method toevaluate antioxidant activity” by W. Brand-Williams et al, published inLWT—Food Science and Technology, Volume 28, Issue 1, 1995, pp 25-30).

Superoxide Scavenging Assay

Superoxide scavenging assay protocol was adapted from “Rapid MicroplateAssay for Superoxide Scavenging Efficiency” in Journal of NeuroscienceMethods 97 (2000) pp. 139-144.

Example 27 Preparation of Cosmetic Botanical Ingredient 601 Derived fromRed Clover (Trifolium pratense) Cell Serum Fractions

The process for preparing Cosmetic Botanical Ingredient 601 wasidentical to the process described in Example 1 with regard to CosmeticBotanical Ingredient 101, with the variations noted below. Fresh stem,flower and leaf tissue of red clover (Trifolium pratense) was used asthe plant biomass starting material. The level of dry matter in thefresh red clover plant biomass was calculated to be 15.16 percent,requiring harvesting of approximately 560 kg of fresh red clover plantbiomass to yield 100 kg of dry matter. The preparation resulted in theproduction of 15.36 kg of Dry Matter yield (or approximately 300 liters)of Cosmetic Botanical Ingredient 601.

Example 28 Product Specifications of Cosmetic Botanical Ingredient 601Derived from Red Clover (Trifolium pratense) Cell Serum Fractions

Cosmetic Botanical Ingredient 601 was prepared according to the processdescribed above in Example 27.

Analyses of Cosmetic Botanical Ingredient 601 were conducted todetermine its various physico-chemical, microbial, and bioactivitycharacteristics, as described below. Cosmetic Botanical Ingredient 601is a clear liquid, which has a yellow-reddish color and a characteristicodor. No solvent (i.e., glycol, oil, or water) was added to the carriermedium.

Table 38 summarizes the Physical, chemical and organolaepticcharacteristics of Cosmetic Botanical Ingredient 601.

TABLE 38 Physical, chemical and organolaeptic characteristics ofCosmetic Botanical Ingredient 601 Characteristics Description/RangeAppearance Clear Yellow-Reddish Liquid Odor Characteristic Solubility inwater Soluble in any ratio Color (Gardner scale) 5-7 Dry matter (%)4.5-6.1 Refractive index (nD) 1.3440-1.3460 pH 3.8-4.2 Osmolality(mOsm/kg) 780-910 UV max, nm 350-358 Total Plate Count (CFU/gm) <10Mold/Yeast (CFU/gm) <10 E. coli (CFU/gm) Negative/10 gm Salmonella sp.(CFU/gm) Negative/10 gm Staphylococcus aureus (CFU/gm) Negative/10 gmPseudomonas sp. (CFU/gm) Negative/10 gm

Cosmetic Botanical Ingredient 601 contained 0.101-0.104% of nitrogendetermined by Kjeldahl method that indicates that it is substantiallyprotein-free

Proteins, including those in plants, can cause protein contactdermatitis in sensitive individuals. Shortly after contact with thecausative proteinacous material, such individuals can experiencesymptoms such as acute urticarial or vesicular eruption on the skin,often accompanied by pruritus, burning, and/or stinging. (V. Janssens,et al., “Protein contact dermatitis: myth or reality?”, British Journalof Dermatology 1995; 132: 1-6).

Thus, it is highly desirable that skin care materials contain as littleproteins as possible. As used herein, “substantially free of proteins”means less than 1% (from 0% to 1%) total protein content using theKjeldahl method.

Microbial analyses demonstrated that Cosmetic Botanical Ingredient 601satisfies the cosmetic industry requirements for cosmetic ingredientswith regard to CFUs and absence of pathogens. Cosmetic BotanicalIngredient 601 was determined to be stable (i.e., maintaining physicaland chemical integrity) for at least 12-18 months while stored at atemperature of between 15 and 25° C. in a closed container protectedfrom light. Cosmetic Botanical Ingredient 601 is a biodegradableingredient.

Example 29 Summary of Activity and Assay Data Regarding CosmeticBotanical Ingredient 601 Red Clover (Trifolium pratense) Cell SerumFractions

According to mushroom tyrosinase inhibitor efficacy testing Red Clover

Serum Fraction can inhibit tyrosinase, achieving IC₅₀ values about 0.02%w/v.

According to superoxide scavenging assay (protocol adapted from “RapidMicroplate Assay for Superoxide Scavenging Efficiency” in Journal ofNeuroscience Methods 97 (2000) pp. 139-144) Red Clover Serum Fraction iscapable of scavenging superoxide, achieving IC₅₀ of about 0.03% w/v.

According to DPPH free radical scavenging assay (protocol based on “Useof a free radical method to evaluate antioxidant activity” in LWT—FoodScience and Technology, Volume 28, Issue 1, 1995, pp 25-30) Red CloverSerum Fraction is capable of quenching DPPH, with about 9.5 units dryweight of the material required to completely quench 1 unit weight DPPH.

According to Oxygen Radical Absorbance Capacity assay (protocol based on“Performing Oxygen Radical Absorbance Capacity (ORAC) Assays withSynergy HT Multi-Detection Microplate Reader” Application Note fromBioTek Instruments) Red Clover Serum Fraction can serve as anantioxidant, with about 4.4 units dry weight of the material required toprovide ORAC effect equal to 1 unit weight of (R)-Trolox methyl ether.

Example 30 Preparation of Cosmetic Botanical Ingredient 701 Derived fromLotus (Nelumbo nucifera) Cell Serum Fractions

The process for preparing Cosmetic Botanical Ingredient 701 wasidentical to the process described in Example 1 with regard to CosmeticBotanical Ingredient 101, with the variations noted below. Fresh stem,flower and leaf tissue of Lotus (Nelumbo nucifera) was used as the plantbiomass starting material. The level of dry matter in the fresh Lotus(Nelumbo nucifera) plant biomass was calculated to be 20.7 percent,requiring harvesting of approximately 483 kg of Lotus (Nelumbo nucifera)plant biomass to yield 100 kg of dry matter. The preparation resulted inthe production of approximately 13 kg Dry Matter yield (or approximately262 liters) of Cosmetic Botanical Ingredient 701.

Example 31 Product Specifications of Cosmetic Botanical Ingredient 701Derived from Lotus (Nelumbo nucifera) Cell Serum Fractions

Cosmetic Botanical Ingredient 701 was prepared according to the processdescribed above in Example 30.

Analyses of Cosmetic Botanical Ingredient 701 were conducted todetermine its various physico-chemical, microbial, and bioactivitycharacteristics, as described below. Cosmetic Botanical Ingredient 701is a clear liquid, which has a yellow color and a characteristic odor.No solvent (i.e., glycol, oil, or water) was added to the carriermedium.

Table 39 summarizes the Physical, chemical and organolaepticcharacteristics of Cosmetic Botanical Ingredient 701.

TABLE 39 Physical, chemical and organolaeptic characteristics ofCosmetic Botanical Ingredient 701 Characteristics Description/RangeAppearance Clear Yellow Liquid Odor Characteristic Solubility in waterSoluble in any ratio Color (Gardner scale) 5-6 Dry matter (%) 4.2-5.5Refractive index (nD) 1.3370-1.3450 pH 3.9-4.4 Osmolality (mOsm/kg)460-610 UV max, nm 261-269 Total Plate Count (CFU/gm) <10 Mold/Yeast(CFU/gm) <10 E. coli (CFU/gm) Negative/10 gm Salmonella sp. (CFU/gm)Negative/10 gm Staphylococcus aureus (CFU/gm) Negative/10 gm Pseudomonassp. (CFU/gm) Negative/10 gm

Cosmetic Botanical Ingredient 701 contains 0.166-0.167% of nitrogendetermined by Kjeldahl method that indicates that it is substantiallyprotein-free.

Proteins, including those in plants, can cause protein contactdermatitis in sensitive individuals. Shortly after contact with thecausative proteinacous material, such individuals can experiencesymptoms such as acute urticarial or vesicular eruption on the skin,often accompanied by pruritus, burning, and/or stinging. (V. Janssens,et al., “Protein contact dermatitis: myth or reality?”, British Journalof Dermatology 1995; 132: 1-6).

Thus, it is highly desirable that skin care materials contain as littleproteins as possible. As used herein, “substantially free of proteins”means less than 1% (from 0% to 1%) total protein content using theKjeldahl method.

Microbial analyses demonstrated that Cosmetic Botanical Ingredient 701satisfies the cosmetic industry requirements for cosmetic ingredientswith regard to CFUs and absence of pathogens Cosmetic BotanicalIngredient 701 was determined to be stable (i.e., maintaining physicaland chemical integrity) for at least 12-18 months while stored at atemperature of between 15 and 25° C. in a closed container protectedfrom light.

Cosmetic Botanical Ingredient 701 is a biodegradable ingredient.

Example 32 Summary of Activity and Assay Data Regarding Lotus (NelumboNucifera) Serum Fractions

According to LDH release cytotoxicity method (MB Research protocol701-01) study completed by MB Research, Lotus Serum Fraction does notshow cytotoxicity in concentration ranges up to 10% of test article byvolume in cell culture medium.

According to MatTek MelanoDerm Assay (MB Research protocol 750-01) LotusSerum Fraction at concentration 5% v/v or 0.19% w/v in cell culturemedium can produce a lightening/whitening effect inkeratinocyte/melanocyte cell culture model as determined byapproximately 30% decrease in melanin levels below negative control anddefinite lightening of the tissue noticeable with a naked eye.

According to trypsin inhibitor efficacy testing performed Lotus SerumFraction is capable of inhibiting trypsin, with IC₅₀ calculated as about0.04% w/v.

According to superoxide scavenging assay (protocol adapted from “RapidMicroplate Assay for Superoxide Scavenging Efficiency” in Journal ofNeuroscience Methods 97 (2000) pp. 139-144) testing Lotus Serum Fractionis capable of scavenging superoxide, achieving IC₅₀ of about 0.016% w/v.

According to DPPH free radical scavenging assay (protocol based on “Useof a free radical method to evaluate antioxidant activity” in LWT—FoodScience and Technology, Volume 28, Issue 1, 1995, pp 25-30) Lotus SerumFraction is capable of quenching DPPH, with about 2.5 units dry weightof the material required to completely quench 1 unit weight DPPH.

According to Oxygen Radical Absorbance Capacity assay (protocol based on“Performing Oxygen Radical Absorbance Capacity (ORAC) Assays withSynergy HT Multi-Detection Microplate Reader” Application Note fromBioTek Instruments) Lotus Serum Fraction can serve as an antioxidant,with about 1.7 units dry weight of the material required to provide ORACeffect equal to 1 unit weight of (R)-Trolox methyl ether.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A bioactive botanical cosmetic composition comprising: a cell serum fraction filtrate derived from cell juice extracted from a fresh plant biomass, said cell serum fraction filtrate having antioxidant activity, cell growth stimulation activity, and/or both antioxidant and cell growth stimulation activities, wherein said fresh plant biomass is from a plant source selected from the group consisting of lotus (Nelumbo nucifera) and red clover (Trifolium pratense); and a stabilizing agent, wherein said cell growth stimulation activity is due to stimulation of proliferation of at least one type of cell, wherein said cell serum fraction filtrate is derived from the cell juice according to the following steps: providing the plant cell juice, said plant cell juice having been extracted from the fresh plant biomass; treating the plant cell juice under conditions effective to separate the plant cell juice into a membrane fraction and a cell juice supernatant; processing the cell juice supernatant under conditions effective to separate the cell juice supernatant into a cytoplasmic fraction and a cell serum fraction; and refining the cell serum fraction under conditions effective to yield the cell serum fraction filtrate, wherein said refining comprises subjecting the cell serum fraction to a temperature treatment step to yield a coagulated cell serum fraction, and clarifying the coagulated cell serum fraction to yield the cell serum fraction filtrate.
 2. The bioactive botanical cosmetic composition according to claim 1, wherein said stabilizing agent is selected from the group consisting of a preservative and an antioxidant.
 3. The bioactive botanical cosmetic composition according to claim 2, wherein said preservative is selected from the group consisting of potassium sorbate, sodium benzoate, sodium methyl paraben, and citric acid.
 4. The bioactive botanical cosmetic composition according to claim 2, wherein said antioxidant is sodium metabisulfite.
 5. The bioactive botanical cosmetic composition according to claim 2, wherein said antioxidant activity is selected from the group consisting of superoxide scavenging activity and neutrophil respiratory burst inhibitory activity.
 6. The bioactive botanical cosmetic composition according to claim 1, wherein said cell serum fraction filtrate comprises between about 1 and about 10 weight percent of said bioactive botanical cosmetic composition.
 7. The bioactive botanical cosmetic composition according to claim 6, wherein said bioactive botanical cosmetic composition has a superoxide scavenging potency range from an ICR₅₀ value of between about 50 and about 190 μg of dry matter/ml, wherein said ICR₅₀ value represents the concentration of dry matter contained in the cell serum fraction filtrate required to inhibit 50 percent of cytochrome c reduction.
 8. The bioactive botanical cosmetic composition according to claim 1, wherein said cell serum fraction filtrate has a cell growth stimulation potency ranging from between about 1.0 and 125 μg of dry matter/ml.
 9. The bioactive botanical cosmetic composition according to claim 8, wherein said cell serum fraction filtrate has an NRU value of between about 110 and 190 percent, wherein said NRU value represents cell viability.
 10. The bioactive botanical cosmetic composition according to claim 1, wherein said bioactive botanical cosmetic composition has an ability to cause biphasic modulation of respiratory bursts from phorbol myristate acetate-stimulated neutrophils, in that said composition inhibits said respiratory bursts at between about 1.0 and 5.0 μg dry material/ml and stimulates said respiratory bursts at between about 20 and 180 μg dry material/ml.
 11. The bioactive botanical cosmetic composition according to claim 1, wherein said bioactive botanical cosmetic composition comprises characteristics substantially the same as those set forth in Table 38 or Table
 39. 12. A bioactive botanical cosmetic formulation, suitable for topical application to a mammal, comprising a cosmetically acceptable carrier and a cosmetically effective amount of the bioactive botanical cosmetic composition according to claim
 1. 13. The cosmetic formulation according to claim 12, wherein the cosmetically acceptable carrier is selected from the group consisting of a hydrophilic cream base, a hydrophilic lotion base, a hydrophilic surfactant base, a hydrophobic cream base, a hydrophobic lotion base, and a hydrophobic surfactant base.
 14. The cosmetic formulation according to claim 12, wherein said bioactive botanical cosmetic composition is present in an amount ranging from between about 0.001 percent and about 95 percent of the total weight of the cosmetic formulation.
 15. The bioactive botanical cosmetic composition according to claim 1, wherein said plant source is lotus (Nelumbo nucifera).
 16. The bioactive botanical cosmetic composition according to claim 1, wherein said plant source is red clover (Trifolium pratense).
 17. The cosmetic formulation according to claim 12, wherein said plant source is lotus (Nelumbo nucifera).
 18. The cosmetic formulation according to claim 12, wherein said plant source is red clover (Trifolium pratense).
 19. A method for preparing a bioactive botanical cosmetic composition, said method comprising: providing a plant cell juice, said plant cell juice having been extracted from a fresh plant biomass, wherein said fresh plant biomass is from a plant source selected from the group consisting of lotus (Nelumbo nucifera) and red clover (Trifolium pratense); treating the plant cell juice under conditions effective to separate the plant cell juice into a membrane fraction and a cell juice supernatant; processing the cell juice supernatant under conditions effective to separate the cell juice supernatant into a cytoplasmic fraction and a cell serum fraction; refining the cell serum fraction under conditions effective to yield a cell serum fraction filtrate having antioxidant activity, cell growth stimulation activity, and/or both antioxidant and cell growth stimulation activities, wherein said refining comprises subjecting the cell serum fraction to a temperature treatment step to yield a coagulated cell serum fraction, and clarifying the coagulated cell serum fraction to yield the cell serum fraction filtrate; and stabilizing the cell serum fraction filtrate under conditions effective to yield a stable bioactive botanical cosmetic composition exhibiting said antioxidant activity, cell growth stimulation activity, or both antioxidant and cell growth stimulation activities.
 20. The method according to claim 19, wherein said treating comprises: coagulating the plant cell juice to yield a coagulated cell juice mixture, and separating the coagulated cell juice to a membrane fraction and a cell juice supernatant.
 21. The method according to claim 20, wherein said coagulating comprises: destabilizing the plant cell juice to yield a coagulated cell juice mixture, wherein said destabilizing is achieved by heat treatment, electro-membrane treatment, chemical treatment, and/or their combination.
 22. The method according to claim 21, wherein said heat treatment comprises: heating the plant cell juice to a heat treatment temperature required to induce coagulation of the membrane fraction, and cooling the heated cell juice to a temperature effective to permit quantitative separation of said membrane fraction from said cell juice supernatant.
 23. The method according to claim 22, wherein said heating is carried out at 45 to 70 degrees Celsius.
 24. The method according to claim 22, wherein said cooling is carried out at 30 to 45 degrees Celsius.
 25. The method according to claim 20, wherein said separating is carried out by filtration or centrifuigation.
 26. The method according to claim 19, wherein said processing comprises: subjecting the cell juice supernatant to a cytoplasmic fraction precipitation step to yield a cytoplasm/cell serum mixture comprising the cytoplasmic fraction and the cell serum fraction, and separating the cytoplasmic fraction from the cell serum fraction.
 27. The method according to claim 26, wherein said cytoplasmic fraction precipitation step is carried out by isoelectric titration, electrodialysis, and/or their combination.
 28. The method according to claim 27, wherein said isoelectric titration comprises: adjusting the pH of the cell juice supernatant to between about 2.5 and 6.5.
 29. The method according to claim 26, wherein said separating is carried out by filtration or centrifuigation.
 30. The method according to claim 19, wherein said temperature treatment step comprises: heating the cell serum fraction to a heating temperature required to induce coagulation within the cell serum fraction, and cooling the cell serum fraction to a temperature effective to allow further quantitative separation of said cell serum fraction filtrate.
 31. The method according to claim 30, wherein said heating temperature is at 80 to 95 degrees Celsius.
 32. The method according to claim 30, wherein said cooling is to a temperature of at least as low as about 15 degrees Celsius.
 33. The method according to claim 19, wherein said clarifying is carried out by filtration or centrifugation.
 34. The method according to claim 33, wherein said filtration comprises: vacuum filtrating the coagulated cell serum fraction to yield said cell serum fraction filtrate.
 35. The method according to claim 19, further comprising: adjusting the pH of the cell serum fraction to about 3.0 to 4.0 immediately prior to said refining.
 36. The method according to claim 19, wherein said stabilizing comprises: incubating said cell serum fraction filtrate in a mixture of at least one preservative and at least one antioxidant to yield the stable bioactive botanical cosmetic composition.
 37. The method according to claim 36, wherein said preservative is selected from the group consisting of potassium sorbate, sodium benzoate, sodium methyl paraben, and citric acid.
 38. The method according to claim 36, wherein said antioxidant is sodium metabisulfite.
 39. The method according to claim 19, wherein said plant source is lotus (Nelumbo nucifera).
 40. The method according to claim 19, wherein said plant source is red clover (Trifolium pratense).
 41. A stable bioactive botanical cosmetic composition made by the method according to claim
 19. 